Humanized collagen antibodies and related methods

ABSTRACT

The invention provides a grafted antibody, or functional fragment thereof, comprising one or more complementarity determining regions (CDRs) having at least one amino acid substitution in one or more CDRs of a heavy chain CDR, where the grafted antibody or functional fragment thereof has specific binding activity for a cryptic collagen epitope. The invention also provides methods of using an antibody having specific binding activity for a cryptic collagen epitope, including methods of inhibiting angiogenesis, tumor growth, and metastasis.

BACKGROUND OF THE INVENTION

The present invention relates generally to immunology and morespecifically to humanized antibodies and uses thereof.

The extracellular matrix (ECM) plays a fundamental role in theregulation of normal and pathological processes. The most abundantlyexpressed component found in the ECM is collagen. Triple helicalcollagen is known to be highly resistant to proteolytic cleavage exceptby members of the matrix metalloproteinase (MMP) family of enzymes.

Angiogenesis and tumor growth depend on cellular interactions with theextracellular matrix. During angiogenesis and tumor invasion, bothendothelial cells as well as tumor cells proteolytically remodel theirextracellular microenvironment. The invasive cells then interact withthis newly remodeled extracellular matrix followed by migration andinvasion. To this end, a major component of the basement membranesurrounding blood vessels is collagen-IV. Moreover, collagen-I is themajor component of the interestitial matrix.

One of the major detrimental consequences of the progression of canceris metastasis beyond the site of the primary tumor. Such metastasisoften requires more aggressive therapies, and once metastasis hasoccurred, the prognosis for survival of a cancer patient decreasesdramatically.

The growth of all solid tumors requires new blood vessel growth forcontinued expansion of the tumors, particularly beyond a minimal size.Because angiogenesis is required for tumor growth, inhibitingangiogenesis is one approach to inhibiting tumor growth. It is thereforedesirable to identify molecules that can target angiogenic vasculature.Particularly attractive molecules for targeting angiogenic vasculatureare antibodies that can bind specifically to angiogenic vasculature.However, since most antibodies are developed in non-human animals suchas mice, these antibodies often have undesirable immunogenic activitythat limits their effectiveness for human therapy.

One approach to overcoming the detrimental properties of non-humanantibodies is to humanize the antibodies by using human antibodyframework region sequences spliced together with the binding domainsthat confer binding specificity. However, grafting of these bindingdomains, referred to as complementarity determining regions (CDRs), intohuman frameworks has often resulted in the loss of binding affinity.

Thus, there exists a need to identify antibodies specific for angiogenicvasculature and to humanize and optimize the antibodies for therapeuticpurposes. The following invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The invention provides a grafted antibody, or functional fragmentthereof, comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR, where the grafted antibody or functional fragmentthereof has specific binding activity for a cryptic collagen epitope.The invention also provides methods of using an antibody having specificbinding activity for a cryptic collagen epitope, including methods ofinhibiting angiogenesis, tumor growth, and metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences of primers used to clone nucleic acidsencoding HUIV26 and HUI77 antibodies. FIG. 1A shows a set of 5′ primersfor the signal peptide of mouse antibody light chain (SEQ ID NOS:184-192). FIG. 1B shows a set of 5′ primers for the signal peptide ofmouse antibody heavy chain (SEQ ID NOS: 193-211). FIG. 1C shows a set ofprimers for the constant region of mouse heavy and light chains. Primer2650 (SEQ ID NO:212) is the 3′ primer for mouse kappa light chainconstant region (amino acids 123-115). Primer 2656 (SEQ ID NO:213) isthe 3′ primer for mouse IgM CH1 region (amino acids 121-114). Primer2706 (SEQ ID NO:214) is the 3′ primer for mouse IgM CH1 region (aminoacids 131-124).

FIG. 2 shows the sequence of the variable region of anti-crypticcollagen site antibody HUIV26. FIG. 2A shows the nucleotide sequence ofHUIV26 variable region light chain (SEQ ID NO:1). FIG. 2B shows thenucleotide sequence of HUIV26 variable region heavy chain (SEQ ID NO:3).FIG. 2C shows an alignment of the amino acid sequence of HUIV26 lightchain (V_(K)) domain of HUIV26 (SEQ ID NO:2) with a human variableregion fusion, VKIV/JK2 (SEQ ID NO:6) and an alignment of HUIV26 heavychain (V_(H)) domain (SEQ ID NO:4) with a human variable region fusionVHIII/JH6 (SEQ ID NO:8), with CDRs underlined. Amino acids in theframework region that differ between the aligned sequences are indicatedby lines.

FIG. 3 shows the sequence of the variable region of anti-crypticcollagen site antibody HUI77. FIG. 3A shows the nucleotide sequence ofHUI77 variable region light chain (SEQ ID NO:9). FIG. 3B shows thenucleotide sequence of HUI77 variable region heavy chain (SEQ ID NO:11).FIG. 3C shows an alignment of the amino acid sequence of HUI77 lightchain (V_(K)) domain of HUI77 (SEQ ID NO:10) with a human variableregion fusion, VKII/JK1 (SEQ ID NO:14) and an alignment of HUI77 heavychain (V_(H)) domain (SEQ ID NO:12) with a human variable region fusionVHIII/JH6 (SEQ ID NO:16), with CDRs underlined. Amino acids in theframwork region that differ between the aligned sequences are indicatedby lines. FIG. 3D shows an alignment of the nucleotide sequence of HUI77variable region with the sequence of the human framework fusion of DPK13and JK1 (SEQ ID NO:17).

FIG. 4 shows beneficial CDR mutations for anti-cryptic collagen siteantibody HUIV26. FIG. 4A shows a set of primers used to generate randommutations in LCDR3 and HCDR3 of HUIV26 (HUIV26 LCDR3 primers, SEQ IDNOS:224-232; HUIV26 HCDR3 primers, SEQ ID NOS:233-243). FIG. 4B shows aset of primers used to generate random mutations in LCDR1a (SEQ IDNOS:266-273), LCDR1b (SEQ ID NOS:274-282), LCDR2 (SEQ ID NOS:283-289),HCDR1 (SEQ ID NOS:290-294), HCDR2a (SEQ ID NOS:295-303) and HCDR2b (SEQID NOS:304-311) of HUIV26. FIG. 4C shows beneficial CDR mutations of theHUIV26 antibody.

FIG. 5 shows beneficial CDR mutations for anti-cryptic collagen siteantibody HUI77. FIG. 5A shows a set of primers used to generate randommutations in LCDR3 (SEQ ID NOS:359-367) and HCDR3 (SEQ ID NOS:368-380)of HUI77. FIG. 5B shows a set of primers used to generate randommutations in LCDR1a (SEQ ID NOS:312-319), LCDR1b (SEQ ID NOS:320-327),LCDR2 (SEQ ID NOS:328-334), HCDR1 (SEQ ID NOS:335-341), HCDR2a (SEQ IDNOS:342-349) and HCDR2b (SEQ ID NOS:350-357) of HUI77. FIG. 5C showsbeneficial CDR mutations of the HUI77 antibody.

FIG. 6 shows mutations in combinatorial variants of the HUIV26 antibody.The position of amino acids are shown, with mutations different thanwild type shown in bold. The relative activity of combinatorial variantsis shown as “SPEK_(on)” and “SPEK_(off)” (last column). Primers used tocreate the combinatorial libraries are also shown (SEQ ID NOS:163-173).

FIG. 7 shows mutations in combinatorial variants of the HUI77 antibody.The position of amino acids are shown, with mutations different thanwild type shown in bold. The relative activity of combinatorial variantsis shown as “SPEK_(on”) and “SPEK_(off)” (last column). Primers used tocreate the combinatorial libraries are also shown (SEQ ID NOS:174-183).

FIG. 8 shows the activity and specificity of HUIV26 variants. Thebinding of purified Fabs of IX-IV26, containing wild type HUIV26 CDRs,and the HUIV26 variants 2D4H1-C3 and DhuG5 is shown for denaturedcollagen IV (FIG. 8A), denatured collagen I (FIG. 8B) and nativecollagen IV (FIG. 8C).

FIG. 9 shows the activity and specificity of HUI77 variants. The bindingof purified Fabs of IX-I77, containing wild type HUI77 CDRs, and HUI77variants Qh2b-B7 and QhuD9 is shown for denatured collagen I (FIG. 9A),denatured collagen IV (FIG. 9B) and native collagen I (FIG. 9C).

FIG. 10 shows the binding activity of the HUIV26 variant DhuH8. Thebinding activity of the Fab form and the IgG form of the antibody todenatured (d-IV) and native (n-IV) human collagen IV is shown.

FIG. 11 shows the effect of the HUI77 variant QH2b on B16 melanoma cellproliferation. B16 melanoma cells grown in culture were not treated(control; squares) or treated with the IgG form of the QH2b variant(circles).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides antibodies specific for a cryptic collagen site,which is exposed during angiogenesis and tumor cell invasion throughcollagenous tissue and thus serves as an antibody that can targetangiogenic vasculature. The antibodies are optimized for bindingactivity to a cryptic collagen site. The antibodies can be used totarget angiogenic vasculature for diagnostic or therapeutic purposes.The antibodies can also be used to inhibit tumor growth.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.These particular regions have been described by Kabat et al., J. Biol.Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and HumanServices, “Sequences of proteins of immunological interest” (1991); byChothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues which encompass the CDRs as defined by each of the abovecited references are set forth below in Table 1 as a comparison.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally between about 100-120 amino acidsin length but is intended to reference only those amino acids outside ofthe CDRs. As used herein, the term “framework region” is intended tomean each domain of the framework that is separated by the CDRs.

As used herein,the term “donor” is intended to mean a parent antibodymolecule or fragment thereof from which a portion is derived from, givento or contributes to another antibody molecule or fragment thereof so asto confer either a structural or functional characteristic of the parentmolecule onto the receiving molecule. For the specific example of CDRgrafting, the parent molecule from which the grafted CDRs are derived isa donor molecule. The donor CDRs confer binding affinity of the parentmolecule onto the receiving molecule. The donor molecule can be adifferent species or the same species as the receiving molecule. If thedonor and receiving molecules are of the same species, it is understoodthat it is sufficient that the donor is a separate and distinct moleculefrom the receiving molecule.

As used herein, the term “acceptor” is intended to mean an antibodymolecule or fragment thereof which is to receive the donated portionfrom the parent or donor antibody molecule or fragment thereof. Anacceptor antibody molecule or fragment thereof is therefore impartedwith the structural or functional characteristic of the donated portionof the parent molecule. For the specific example of CDR grafting, anacceptor molecule, including framework and/or other antibody fragments,is the receiving molecule into which the CDRs are grafted. The acceptorantibody molecule or fragment is imparted with the binding affinity ofthe donor CDRs or parent molecule. As with a donor molecule, it isunderstood that an acceptor molecule can be the same or a differentspecies as the donor.

A “variable region” when used in reference to an antibody or a heavy orlight chain thereof is intended to mean the amino terminal portion of anantibody which confers antigen binding onto the molecule and which isnot the constant region. The term is intended to include functionalfragments thereof which maintain some of all of the binding function ofthe whole variable region. Therefore, the term “heteromeric variableregion binding fragments” is intended to mean at least one heavy chainvariable region and at least one light chain variable regions orfunctional fragments thereof assembled into a heteromeric complex.Heteromeric variable region binding fragments include, for example,functional fragments such as Fab, F(ab)₂, Fv, single chain Fv (scFv) andthe like. Such functional fragments are well known to those skilled inthe art. Accordingly, the use of these terms in describing functionalfragments of a heteromeric variable region is intended to correspond tothe definitions well known to those skilled in the art. Such terms aredescribed in, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biologyand Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.),New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics,22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515(1989); and in Day, E. D., Advanced Immunochemistry, Second Ed.,Wiley-Liss, Inc., New York, N.Y. (1990).

As used herein, the term “population” is intended to refer to a group oftwo or more different molecules. A population can be as large as thenumber of individual molecules currently available to the user or ableto be made by one skilled in the art. Populations can be as small as 2-4molecules or as large as 10¹³ molecules. Generally, a population willcontain two or more, three or more, five or more, nine or more, ten ormore, twelve or more, fifteen or more, or twenty or more differentmolecules. A population can also contain tens or hundreds of differentmolecules or even thousands of different molecules. For example, apopulation can contain about 20 to about 100,000 different molecules ormore, for example about 25 or more, 30 or more, 40 or more, 50 or more,75 or more, 100 or more, 150 or more, 200 or more, 300 or more, 500 ormore, or 1000 or more different molecules, and can contain 10,000,100,000 or even 1×10⁶ or more different molecules. Those skilled in theart will know what size and diversity of a population is suitable for aparticular application.

As used herein, the term “altered” when used in reference to an antibodyvariable region is intended to mean a heavy or light chain variableregion that contains one or more amino acid changes in a frameworkregion, a CDR or both compared to the parent amino acid sequence at thesame position. Where an altered variable region is derived from orcomposed of donor and acceptor regions, the changed amino acid residueswithin the altered species are to be compared to their respective aminoacid positions within the parent donor and acceptor regions.

As used herein, the term “nucleic acid” or “nucleic acids” is intendedto mean a single- or double-stranded DNA or RNA molecule. A nucleic acidmolecule of the invention can be of linear, circular or branchedconfiguration, and can represent either the sense or antisense strand,or both, of a nucleic acid molecule. The term also is intended toinclude nucleic acid molecules of both synthetic and natural origin. Anucleic acid molecule of natural origin can be derived from any animal,such as a human, non-human primate, mouse, rat, rabbit, bovine, porcine,ovine, canine, feline, or amphibian, or from a lower eukaryote, such asDrosophila, C. elegans, yeast, and the like. A synthetic nucleic acidincludes, for example, chemical and enzymatic synthesis. The term“nucleic acid” or “nucleic acids” is similarly intended to includeanalogues of natural nucleotides which have similar functionalproperties as the referenced nucleic acid and which can be utilized in amanner similar to naturally occurring nucleotides and nucleosides.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. An antibody useful in the invention, orantigen binding fragment of such an antibody, is characterized by havingspecific binding activity for a polypeptide or a peptide portion thereofof at least about 1×10⁵ M⁻¹. Thus, Fab, F(ab′)₂, Fd, Fv, single chain Fv(scFv) fragments of an antibody and the like, which retain specificbinding activity for a polypeptide, are included within the definitionof an antibody. Specific binding activity of an antibody for apolypeptide can be readily determined by one skilled in the art, forexample, by comparing the binding activity of an antibody to aparticular polypeptide versus a control polypeptide that is not theparticular polypeptide. Methods of preparing polyclonal or monoclonalantibodies are well known to those skilled in the art (see, for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988)).

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al. (Science 246:1275-1281 (1989)). These and other methods of makingfunctional antibodies are well known to those skilled in the art (Winterand Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al.,Protein Engineering: A practical approach (IRL Press 1992); Borrabeck,Antibody Engineering, 2d ed. (Oxford University Press 1995)).

As used herein, specific binding means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity, for example, an antibodythat binds a distinct epitope or antigen. Specificity of binding alsocan be determined, for example, by competition with a control molecule,for example, competition with an excess of the same molecule. In thiscase, specific binding is indicated if the binding of a molecule iscompetitively inhibited by itself. Thus, specific binding between anantibody and antigen is measurably different from a non-specificinteraction and occurs via the antigen binding site of the antibody.

As used herein, selective binding refers to a binding interaction thatis both specific and discriminating between molecules, for example, anantibody that binds to a single molecule or closely related molecules.For example, an antibody can exhibit specificity for an antigen that canbe both specific and selective for the antigen if the epitope is uniqueto a molecule. Thus, a molecule having selective binding candifferentiate between molecules, as exemplified by an antibody havingspecificity for an epitope unique to one molecule or closely relatedmolecules. Alternatively, an antibody can have specificity for anepitope that is common to many molecules, for example, a carbohydratethat is expressed on a number of molecules. Such an antibody hasspecific binding but is not selective for one molecule or closelyrelated molecules.

As used herein the term “binding affinity” is intended to mean thestrength of a binding interaction and includes both the actual bindingaffinity as well as the apparent binding affinity. The actual bindingaffinity is a ratio of the association rate over the disassociationrate. Therefore, conferring or optimizing binding affinity includesaltering either or both of these components to achieve the desired levelof binding affinity. The apparent affinity can include, for example, theavidity of the interaction. For example, a bivalent heteromeric variableregion binding fragment can exhibit altered or optimized bindingaffinity due to its valency.

As used herein, the term “substantially the same” when used in referenceto binding affinity is intended to mean similar or identical bindingaffinities where one molecule has a binding affinity that is similar toanother molecule within the experimental variability of the affinitymeasurement. The experimental variability of the binding affinitymeasurement is dependent upon the specific assay used and is known tothose skilled in the art.

As used herein, the term “optimizing” when used in reference to avariable region or a functional fragment thereof is intended to meanthat the functional activity of the variable region has been modifiedcompared to the activity of a parent variable region or a donor variableregion, resulting in a desirable change in activity. A variable regionor functional fragment thereof exhibiting optimized activity canexhibit, for example, higher affinity or lower affinity binding, orincreased or decreased association or dissociation rates compared to anunaltered variable region. A variable region or functional fragmentthereof exhibiting optimized activity also can exhibit increasedstability such as increased half-life in a particular organism. Forexample, an antibody activity can be optimized to increase stability bydecreasing susceptibility to proteolysis. An antibody exhibitingoptimized activity also can exhibit lower affinity binding, includingdecreased association rates or increased dissociation rates, if desired.An optimized variable region exhibiting lower affinity binding isuseful, for example, for penetrating a solid tumor. In contrast to ahigher affinity variable region, which would bind to the peripheralregions of the tumor but would be unable to penetrate to the innerregions of the tumor due to its high affinity, a lower affinity variableregion would be advantageous for penetrating the inner regions of thetumor. As with optimization of binding affinities above, optimization ofa catalytic variable region can be, for example, increased or decreasedcatalytic rates, disassociation constants or association constants.

As used herein, a “cryptic collagen site” or “cryptic collagen epitope”refers to an epitope of a collagen molecule that is less accessible tobinding of an antibody, or functional fragment thereof, in nativecollagen than in denatured collagen. An antibody having binding activityfor a cryptic collagen epitope preferentially recognizes denaturedcollagen over native collagen, that is, has a higher binding affinityfor denatured over native collagen. For example, such an antibody canhave at least about a 2-fold or greater preference, that is, at leastabout 2-fold higher binding activity, for denatured collage over nativecollagen, and can exhibit about a 3-fold or greater preference, about a5-fold or greater preference, about a 10-fold or greater preference,about a 15-fold or greater preference, about a 20-fold or greaterpreference, about a 25-fold or greater preference, about a 50-fold orgreater preference, about a 100-fold or greater preference, or even ahigher preference for denatured over native collagen.

Native collagen herein refers to a molecule where three alpha-chains areorganized in a triple helical molecule. Native collagen can be ofdifferent stages of post-translational processing such as pro-collagenand any intermediates in the generation of a mature tissue form ofcollagen, or collagen molecules isolated by limited proteolysis oftissues under conditions where the triple-helical structure of collagenis not disrupted. Thus, native collagen can be an intact collagenmolecule or can contain non-triple-helical sequences flankingtriple-helical regions, so long as the triple-helical is not disrupted.Denatured collagen herein refers to collagen where the triple helix iscompletely or partially disrupted such that a cryptic epitope is madeaccessible. Denaturation of collagen can occur in situ by the action ofproteinases, for example, matrix metalloproteinases, that cleavecollagen within triple helical regions, rendering the resultingfragments of the triple helix unstable. Denaturation of collagen can beinduced in vitro by thermal or chemical denaturation of native collagen.Denatured collagen can also be prepared in vitro by treatment of nativecollagen with proteinases capable of cleaving a triple helicalregion(s), which are commonly referred to as collagenolytic enzymes, attemperatures where the resulting fragments of the triple helix arethermally unstable. Denatured collagen can be obtained by denaturationof native collagens of different stages of post-translational processingor denaturation of native collagen isolated from tissues by limitedproteolysis. One skilled in the art will know a variety of methods forisolation of native collagens and a variety of methods to denature atriple helix that contains a cryptic collagen epitope.

An antibody of the invention can have binding activity for a crypticcollagen epitope that is the same as the respective parental mouseantibody. For example, an antibody of the invention having CDRs derivedfrom HUIV26 can have essentially the same binding specificity as themouse HUIV26 antibody described by Xu et al., Hybridoma 19:375-385(2000); Xu et al., J. Cell Biol. 154:1069-1079 (2001); and WO 00/40597,which is incorporated herein by reference. Similarly, an antibody of theinvention having CDRs derived from HUI77 can have essentially the samebinding specificity as the mouse HUI77 antibody described by Xu et al.,supra, 2000; Xu et al., supra, 2001; and WO 00/40597. Such bindingspecificity can be tested by the methods disclosed herein, for example,by comparing the activity of an antibody of the invention to thecorresponding parental mouse antibody. For example, an antibody of theinvention derived from HUIV26 can be compared to a corresponding mouseantibody having the variable region amino acid sequence shown in FIG. 2C(SEQ ID NOS:2 and 4). Similarly, an antibody of the invention derivedfrom HUI77 can be compared to a corresponding mouse antibody having thevariable region amino acid sequence shown in FIG. 3C (SEQ ID NOS:10 and12). Similar binding specificity can be determined, for example, bycompetitive binding with the corresponding parental antibody. It isunderstood that an antibody of the invention can have essentially thesame specificity as the corresponding parental antibody or can havealtered specificity so long as the antibody has binding activity for acryptic collagen epitope.

The invention provides antibodies having specific binding activity for acryptic collagen epitope. The antibodies contain at least one CDR havingat least one amino acid substitution in a CDR of the antibodies HUIV26and HUI77, which are antibodies that bind to a cryptic collagen site.The invention also provides nucleic acids encoding these antibodies. Theinvention further provides methods using the antibodies.

Highly specific monoclonal antibodies have been developed that recognizea cryptic domain of human collagen, designated HUIV26 and HUI77 (see Xuet al., Hybridoma 19:375-385 (2000); Xu et al., J. Cell Biol.154:1069-1079 (2001); WO 00/40597, which is incorporated herein byreference). Monoclonal antibody HUIV26 recognizes a cryptic domain ofhuman collagen-IV, and HUI77 recognizes a cryptic domain of humancollagen-I and IV that is also common to collagens II, III and V. Thiscryptic domain(s) is less accessible under most normal physiologicalconditions but becomes accessible following proteolytic remodeling ofthe collagen triple helix in vivo. Thus, cryptic collagen epitope(s) canbecome more accessible during invasive cellular processes. Importantly,the cryptic domain(s) defined by these antibodies was shown to beexposed within the basement membrane of tumor associated angiogenicblood vessels from human tumors including, breast, bladder and melanomatumors. However, this cryptic domain was less exposed within the vesselsor normal tissues tested. Therefore, the antibodies HUIV26 and HUI77represent important and specific markers of angiogenic blood vessels.These cryptic domain(s) plays an important role in regulatingangiogenesis and tumor growth since the monoclonal antibodies HUIV26 andHUI77 potently inhibit angiogensis and human tumor growth in the chickembryo, rat and mouse models following systemic administration (Xu etal., supra, 2001). Thus, these monoclonal antibodies and the antibodiesof the invention having specific binding activity for these crypticcollagen site(s) represent a highly potent and effective new therapeuticreagent for the treatment for diseases characterized by aberrantneovascularization.

A nucleic acid sequence of the invention can include a sequence that isthe same or substantially the same as a specifically recited SEQ ID NO.Similarly, an amino acid sequence of the invention can include asequence that is the same or substantially the same as a specificallyrecited SEQ ID NO. As used herein, the term “substantially” or“substantially the same” when used in reference to a nucleotide or aminoacid sequence is intended to mean that the nucleotide or amino acidsequence shows a considerable degree, amount or extent of sequenceidentity when compared to a reference sequence, for example, thesequence of a parent antibody. Such a considerable degree, amount orextent of sequence identity is further considered to be significant andmeaningful and therefore exhibit characteristics which are definitivelyrecognizable or known. Thus, a nucleotide sequence which issubstantially the same nucleotide sequence as a heavy or light chain ofan antibody of the invention, including fragments thereof, refers to asequence which exhibits characteristics that are definitively known orrecognizable as encoding or as being the amino acid sequence as theparent antibody sequence. Minor modifications thereof are included solong as they are recognizable as a parent antibody sequence. Similarly,an amino acid sequence which is substantially the same amino acidsequence as a heavy or light chain of an antibody of the invention, orfunctional fragment thereof, refers to a sequence which exhibitscharacteristics that are definitively known or recognizable asrepresenting the amino acid sequence of parent antibody and minormodifications thereof. When determining whether a nucleotide or aminoacid sequence is substantially the same as a parent antibody,consideration is given to the number of changes relative to the parentantibody together with whether the function is maintained, for example,whether the function of binding to a cryptic collagen site is maintainedfor antibodies of the invention.

Minor modification of these nucleotide sequences and/or amino acids areintended to be included as heavy and light chain encoding nucleic acidsand their functional fragments. Such minor modifications include, forexample, those which do not change the encoded amino acid sequence dueto the degeneracy of the genetic code as well as those which result inonly a conservative substitution of the encoded amino acid sequence.Conservative substitutions of encoded amino acids include, for example,amino acids which belong within the following groups: (1) non-polaramino acids (Gly, Ala, Val, Leu, and Ile); (2) polar neutral amino acids(Cys, Met, Ser, Thr, Asn, and Gln); (3) polar acidic amino acids (Aspand Glu); (4) polar basic amino acids (Lys, Arg and His); and (5)aromatic amino acids (Phe, Trp, Tyr, and His). Other minor modificationsare included within the nucleic acids encoding heavy and light chainpolypeptides of the invention so long as the nucleic acid or encodedpolypeptides retain some or all of their function as described herein.

To generate antibodies of the invention having specific binding activityfor a cryptic collagen epitope, the heavy and light chain variableregions of the antibodies HUIV26 and HUI77 were cloned and sequenced(see Example I and FIGS. 2 and 3). CDRs of the heavy and light chainvariable regions were identified. Exemplary heavy and light chain CDRs,as determined by the numbering of Kabat, are shown in FIGS. 2C and 3C(underlined). Exemplary heavy and light chain CDRs of HUIV26 include,for example, V_(L) CDR1, KSSQSLLNSGNQKNYLA (SEQ ID NO:20); V_(L) CDR2,GASTRES (SEQ ID NO:22); V_(L) CDR3, QNDHSYPYT (SEQ ID NO:24); V_(E)CDR1, GFDFSRYWMS (SEQ ID NO:26); V_(H) CDR2, EINPDSSTINYTPSLKD (SEQ IDNO:28); and V_(H) CDR3, PVDGYYDAMDY (SEQ ID NO:30). Exemplary heavy andlight chain CDRs of HUI77 include, for example, V_(L) CDR1,RSSQSIVHSNGNTYLE (SEQ ID NO:32); V_(L) CDR2, KVSNRFS (SEQ ID NO:34);V_(L) CDR3, FQGSHVPWT (SEQ ID NO:36); V_(H) CDR1, GFSLSTSGMGVG (SEQ IDNO:38); V_(H) CDR2, DIWWDDNKYYNPSLKS (SEQ ID NO:40); and V_(H) CDR3,RANYGNPYYAMDY (SEQ ID NO:42).

Libraries of CDR variants containing single amino acid substitutionswere generated (Example II). The libraries were screened for binding toa cryptic collagen site, and single amino acid mutations havingbeneficial activity were identified. Combinatorial mutants, in which twoor more variant CDRs containing at least one amino acid substitutionrelative to parental HUIV26 or HUI77 CDRs were combined and screened foractivity (Example III). A number of combinatorial mutants havingoptimized activity for binding to a cryptic collagen site wereidentified.

The antibodies of the invention having binding activity for a crypticcollagen epitope. As disclosed herein, the collagen can be denatured byany of a variety of methods so long as an antigenic determinant isexposed that was less accessible in native collagen. Such methodsinclude, for example, proteolytic digestion, heat or thermaldenaturation, chemical denaturation, and the like. One skilled in theart will know a variety of methods suitable for denaturing a collagenmolecule to reveal a cryptic collagen site or epitope. Furthermore, themethod of denaturation can be a combination of two or more denaturationmethods, for example, proteolytic digestion combined with chemicaland/or thermal denaturation. For example, proteolytic digestion can beused to cleave collagen, resulting in a collagen molecule that is moresusceptible to thermal or chemical denaturation. An exemplary proteasethat can be used to denature collagen is matrix metalloproteinase, whichcan be used in vitro and can function in vivo to cleave collagen withintriple helical regions and at body temperature in a mammal.

The invention provides grafted antibodies of the HUIV26 and HUI77antibodies. In one embodiment, the invention provides a grafted antibodyof HUIV26. The grafted antibody, or functional fragment thereof,comprises one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:26, 28 and 30or a light chain CDR selected from the group consisting of SEQ IDNOS:20, 22 and 24, the grafted antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.

In another embodiment, the invention provides a grafted antibody ofHUI77. The grafted antibody, or functional fragment thereof, comprisesone or more complementarity determining regions (CDRs) having at leastone amino acid substitution in one or more CDRs of a heavy chain CDRselected from the group consisting of SEQ ID NOS:38, 40 and 42 or alight chain CDR selected from the group consisting of SEQ ID NOS:32, 34and 36, the grafted antibody or functional fragment thereof havingspecific binding activity for a cryptic collagen epitope.

The invention additionally provides antibodies, or functional fragmentsthereof, containing specifically recited CDRs, where the antibody orfunctional fragment thereof has specific binding activity for a crypticcollagen epitope. Such antibodies include those having at least a singleamino acid substitution and which retain binding activity for a crypticcollagen epitope. Included among such CDR variants are those describedin FIGS. 4 and 5.

Exemplary CDRs of the invention having a single amino acid substitutionin a CDR of HUIV26 include, for example, those described below, in whichthe position of the amino acid mutation in the numbering of Kabat isindicated along with the amino acid substitution from wild type tomutant (wild type-mutant). Such exemplary CDRs include HuIV26 V_(H) CDR131R→H (SEQ ID NO:43); HuIV26 V_(H) CDR1 34M→I (SEQ ID NO:44); HuIV26V_(H) CDR1 35S→T (SEQ ID NO:45); HuIV26 V_(H) CDR1 35S→A (SEQ ID NO:46);HuIV26 V_(H) CDR1 35S→G (SEQ ID NO:47); HuIV26 V_(H) CDR2 57I→A (SEQ IDNO:48); HuIV26 V_(H) CDR2 57I→S (SEQ ID NO:49); HuIV26 V_(H) CDR2 62S→Y(SEQ ID NO:50); HuIV26 V_(H) CDR2 62S→A (SEQ ID NO:51); HuIV26 V_(H)CDR2 62S→H (SEQ ID NO:52); HuIV26 V_(H) CDR2 62S→G (SEQ ID NO:53);HuIV26 V_(H) CDR2 64K→Q (SEQ ID NO:54); HuIV26 V_(H) CDR2 65D→S (SEQ IDNO:55); HuIV26 V_(H) CDR3 97D→P (SEQ ID NO:56); HuIV26 V_(H) CDR3 97D→G(SEQ ID NO:57); HuIV26 V_(H) CDR3 97D→T (SEQ ID NO:58); HuIV26 V_(H)CDR3 97D→A (SEQ ID NO:59); HuIV26 V_(H) CDR3 98G→P (SEQ ID NO:60);HuIV26 V_(H) CDR3 98G→A (SEQ ID NO:61); HuIV26 V_(H) CDR3 98G→H (SEQ IDNO:62); HuIV26 V_(H) CDR3 102Y→P (SEQ ID NO:63); HuIV26 V_(H) CDR3102Y→N (SEQ ID NO:64); HuIV26 V_(L) CDR1 27Q→R (SEQ ID NO:65); HuIV26V_(L) CDR1 27Q→S (SEQ ID NO:66); HuIV26 V_(L) CDR1 27dN→S (SEQ IDNO:67); HuIV26 V_(L) CDR1 27eS→Y (SEQ ID NO:68); HuIV26 V_(L) CDR127eS→W (SEQ ID NO:69); HuIV26 V_(L) CDR1 27eS→H (SEQ ID NO:70); HuIV26V_(L) CDR1 27eS→R (SEQ ID NO:71); HuIV26 V_(L) CDR1 27fG→Y (SEQ IDNO:72); HuIV26 V_(L) CDR1 27fG→R (SEQ ID NO:73); HuIV26 V_(L) CDR127fG→H (SEQ ID NO:74); HuIV26 V_(L) CDR1 27fG→I (SEQ ID NO:75); HuIV26V_(L) CDR1 29Q→K (SEQ ID NO:76); HuIV26 V_(L) CDR3 93S→Q (SEQ ID NO:77);HuIV26 V_(L) CDR3 93S→G (SEQ ID NO:78); HuIV26 V_(L) CDR3 93S→L (SEQ IDNO:79); HuIV26 V_(L) CDR3 93S→A (SEQ ID NO:80); HuIV26 V_(L) CDR3 93S→T(SEQ ID NO:81); HuIV26 V_(L) CDR3 93S→V (SEQ ID NO:82); HuIV26 V_(L)CDR3 94Y→N (SEQ ID NO:83); HuIV26 V_(L) CDR3 94Y→S (SEQ ID NO:84);HuIV26 V_(L) CDR3 94Y→P (SEQ ID NO:85); HuIV26 V_(L) CDR3 94Y→M (SEQ IDNO:86); and HuIV26 V_(L) CDR2 57I→V (SEQ ID NO:162).

Exemplary CDRs of the invention having a single amino acid substitutionin a CDR of HUI77 include, for example, those described below, in whichthe position of the amino acid mutation in the numbering of Kabat isindicated along with the amino acid substitution from wild type tomutant (wild type-mutant). Such exemplary CDRs include HUI77 V_(H) CDR132S→P (SEQ ID NO:87); HUI77 V_(H) CDR1 32S→W (SEQ ID NO:88); HUI77 V_(H)CDR1 35bG→W (SEQ ID NO:89); HUI77 V_(H) CDR1 35bG→L (SEQ ID NO:90);HUI77 V_(H) CDR1 35bG→A (SEQ ID NO:91); HUI77 V_(H) CDR2 59Y→S (SEQ IDNO:92); HUI77 V_(H) CDR2 59Y→A (SEQ ID NO:93); HUI77 V_(H) CDR2 59Y→P(SEQ ID NO:94); HUI77 V_(H) CDR2 64K→P (SEQ ID NO:95); HUI77 V_(H) CDR395R→P (SEQ ID NO:96); HUI77 V_(H) CDR3 95R→Q (SEQ ID NO:97); HUI77 V_(H)CDR3 95R→L (SEQ ID NO:98); HUI77 V_(H) CDR3 95R→T (SEQ ID NO:99); HUI77V_(H) CDR3 95R→V (SEQ ID NO:100); HUI77 V_(H) CDR3 100N→V (SEQ IDNO:101); HUI77 V_(H) CDR3 100N→W (SEQ ID NO:102); HUI77 V_(H) CDR3100eM→Q (SEQ ID NO:103); HUI77 V_(H) CDR3 100eM→N (SEQ ID NO:104); HUI77V_(H) CDR3 100eM→T (SEQ ID NO:105); HUI77 V_(H) CDR3 102Y→K (SEQ IDNO:106); HUI77 V_(L) CDR3 102Y→T (SEQ ID NO:107); HUI77 V_(H) CDR3102Y→M (SEQ ID NO:108); HUI77 V_(H) CDR3 102Y→H (SEQ ID NO:109); HUI77V_(L) CDR1 27cV→P (SEQ ID NO:110); HUI77 V_(L) CDR1 27cV→W (SEQ IDNO:111); HUI77 V_(L) CDR1 27dH→L (SEQ ID NO:112); HUI77 V_(L) CDR127dH→S (SEQ ID NO:113); HUI77 V_(L) CDR1 27eS→W (SEQ ID NO:114); HUI77V_(L) CDR1 28N→Y (SEQ ID NO:115); HUI77 V_(L) CDR1 28N→W (SEQ IDNO:116); HUI77 V_(L) CDR1 30N→Y (SEQ ID NO:117); HUI77 V_(L) CDR1 33L→F(SEQ ID NO:118); HUI77 V_(L) CDR1 33L→V (SEQ ID NO:119); HUI77 V_(L)CDR2 50K→S (SEQ ID NO:120); HUI77 V_(L) CDR2 51V→A (SEQ ID NO:121);HUI77 V_(L) CDR2 53N→S (SEQ ID NO:122); HUI77 V_(L) CDR2 54R→L (SEQ IDNO:123); HUI77 V_(L) CDR2 56S→W (SEQ ID NO:124); HUI77 V_(L) CDR2 56S→F(SEQ ID NO:125); HUI77 V_(L) CDR3 89F→V (SEQ ID NO:126); HUI77 V_(L)CDR3 89F→H (SEQ ID NO:127); HUI77 V_(L) CDR3 90Q→R (SEQ ID NO:128);HUI77 V_(L) CDR3 90Q→W (SEQ ID NO:129); HUI77 V_(L) CDR3 91G→S (SEQ IDNO:130); HUI77 V_(L) CDR3 92S→W (SEQ ID NO:131); HUI77 V_(L) CDR3 92S→E(SEQ ID NO:132); HUI77 V_(L) CDR3 93H→L (SEQ ID NO:133); HUI77 V_(L)CDR3 93H→T (SEQ ID NO:134); HUI77 V_(L) CDR3 93H→S (SEQ ID NO:135);HUI77 V_(L) CDR3 93H→A (SEQ ID NO:136); HUI77 V_(L) CDR3 93H→Q (SEQ IDNO:137); HUI77 V_(L) CDR3 94V→T (SEQ ID NO:138); HUI77 V_(L) CDR3 97T→A(SEQ ID NO:139); HUI77 V_(L) CDR3 97T→R (SEQ ID NO:140); HUI77 V_(L)CDR3 97T→H (SEQ ID NO:141); HUI77 V_(L) CDR3 97T→K (SEQ ID NO:142);HUI77 V_(L) CDR3 97T→I (SEQ ID NO:143); HUI77 V_(H) CDR2 59Y→T (SEQ IDNO:144); HUI77 V_(L) CDR3 94V→F (SEQ ID NO:145); and HUI77 V_(L) CDR128N→Q (SEQ ID NO:146)

In addition to CDRs having single amino acid substitutions, theinvention additionally provides HUIV26 and HUI77 CDRs having two or moreamino acid substitutions. Exemplary CDRs having two or more amino acidsubstitutions in HUIV26 include, for example, HUIV26 V_(H) CDR257I→A/62S→A (SEQ ID NO:154); HUIV26 V_(H) CDR2 57I→A/62S→Y (SEQ IDNO:155); HUIV26 V_(H) CDR2 57I→A/62S→H (SEQ ID NO:156); HUIV26 V_(L)CDR1 27eS→W/27fG→Y (SEQ ID NO:157); HUIV26 V_(L) CDR1 27eS→Y/27fG→Y (SEQID NO:158); HUIV26 V_(L) CDR1 27eS→Y/27fG→H (SEQ ID NO:159); HUIV26V_(L) CDR1 27eS→R/27fG→Y (SEQ ID NO:160); and HUIV26 V_(L) CDR127eS→W/27fG→H (SEQ ID NO:161) (see FIG. 6). Exemplary CDRs having two ormore amino acid substitutions in HUI77 include, for example, HUI77 V_(H)CDR1 32S→P/35bG→W (SEQ ID NO:147); HUI77 V_(H) CDR1 32S→P/35bG→A (SEQ IDNO:148); HUI77 V_(L) CDR1 27dH→S/28N→W (SEQ ID NO:149); HUI77 V_(L) CDR127dH→S/28N→Y (SEQ ID NO:150); HUI77 V_(L) CDR1 27d→HS/28N→Q (SEQ IDNO:151); HUI77 V_(L) CDR1 28N→Q/33L→F (SEQ ID NO:152); HUI77 V_(L) CDR127H→S/28N→W/33L→F (SEQ ID NO:153); and HUI77 V_(L) CDR3 91G→S/94V→F (SEQID NO:358) (see FIG. 7).

The invention provides an antibody having at least one of the abovevariant CDR sequences. It is understood that any combination of HUIV26CDRs can be combined with mutant and/or wild type CDRs to generate anHUIV26 grafted antibody, so long as binding activity to a crypticcollagen site is maintained. Similarly, any combination of HUI77 CDRscan be combined with mutant and/or wild type CDRs to generate a HUI77grafted antibody so long as binding activity to a cryptic collagen siteis maintained. Thus, any combination of single amino acid substitutionscan be combined with other CDR mutants to generate an antibody having atleast two variant CDRs. Furthermore, any single mutation at differentpositions within the same CDR can be combined to generate a CDR having 2or more amino acid substitutions at two or more positions. Any of thesingle or multiple mutations can be combined so long as binding activityto a cryptic collagen site is maintained.

Thus, the invention provides an antibody, or functional fragmentthereof, comprising one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78; SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO: 154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162, the antibody or functional fragment thereof having specificbinding activity for a cryptic collagen epitope.

The invention additionally provides an antibody, or functional fragmentthereof, comprising one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ IDNO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123,SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ IDNO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151,SEQ ID NO:152, SEQ ID NO:153, and SED ID NO:358 the antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.

The invention further provides an antibody, or functional fragmentthereof, comprising a heavy chain polypeptide comprising one or moreCDRs having at least one amino acid substitution in one or more heavychain CDRs, the heavy chain CDRs selected from the group consisting of aheavy chain CDR1 selected from the group consisting of CDRs referencedas SEQ ID NOS:26, 43, 44, 45, 46, and 47; a heavy chain CDR2 selectedfrom the group consisting of CDRs referenced as SEQ ID NOS:28, 48, 49,50, 51, 52, 53, 54, and 55; and a heavy chain CDR3 selected from thegroup consisting of CDRs referenced as SEQ ID NOS:30, 56, 57, 58, 59,60, 61, 62, 63, and 64, the antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.

The invention also provides an antibody, or functional fragment thereof,comprising a light chain polypeptide comprising one or more CDRs havingat least one amino acid substitution in one or more light chain CDRs,the light chain CDRs selected from the group consisting of a light chainCDR1 selected from the group consisting of CDRs referenced as SEQ IDNOS:20, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76; a lightchain CDR2 referenced as SEQ ID NO:22:; and a light chain CDR3 selectedfrom the group consisting of CDRs referenced as SEQ ID NOS:24, 77, 78,79, 80, 81, 82, 83, 84, 85, and 86, the antibody or functional fragmentthereof having specific binding activity for a cryptic collagen epitope.

The invention further provides an antibody, or functional fragmentthereof, comprising a heavy chain polypeptide comprising one or moreCDRs having at least one amino acid substitution in one or more heavychain CDRs, the heavy chain CDRs selected from the group consisting of aheavy chain CDR1 selected from the group consisting of CDRs referencedas SEQ ID NOS:38, 87, 88, 89, 90, 91, 147 and 148; a heavy chain CDR2selected from the group consisting of CDRs referenced as SEQ ID NOS:40,92, 93, 94, 95 and 144; and a heavy chain CDR3 selected from the groupconsisting of CDRs referenced as SEQ ID NOS:42, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108 and 109, the antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.

Additionally provided is an antibody, or functional fragment thereof,comprising a light chain polypeptide comprising one or more CDRs havingat least one amino acid substitution in one or more light chain CDRs,the light chain CDRs selected from the group consisting of a light chainCDR1 selected from the group consisting of CDRs referenced as SEQ IDNOS:32, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 146, 149, 150,151, 152 and 153; a light chain CDR2 referenced as SEQ ID NOS:34, 120,121, 122, 123, 124 and 125; and a light chain CDR3 selected from thegroup consisting of CDRs referenced as SEQ ID NOS:36, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 145 and 358, the antibody or functional fragment thereof havingspecific binding activity for a cryptic collagen epitope.

As described above, an antibody of the invention can be generated fromany combination of the variant and/or wild type CDRs, so long as bindingactivity to a cryptic collagen site is maintained. As disclosed herein,a variety of combinatorial antibodies containing multiple CDRs having atleast a single amino acid substitution were identified having bindingactivity for a cryptic collagen site. In addition to antibodiescontaining any combination of the respective CDRs disclosed herein, thefollowing specific combinations of CDRs are also provided by theinvention.

Exemplary HUIV26 variants include, for example, the followingantibodies:

-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (4.1-2D4).-   An antibody comprises a heavy chain CDR1 referenced as SEQ ID NO:26;    a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3    referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ ID    NO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a light    chain CDR3 referenced as SEQ ID NO:77 (L1b-F11).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:48; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (H2a-G8).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomA2).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:158; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomA4).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:46; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:159; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomB1).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:48; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomD2).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomD3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomD6).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomE3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:46; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomG2).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:162; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:158; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomA7).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:156; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomB10).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:26; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomC8).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomD7).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:46; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:161; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomD11).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:46; a heavy chain CDR2 referenced as SEQ ID NO:156; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:161; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (DcomE11).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:46; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain    CDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as    SEQ ID NO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a    light chain CDR3 referenced as SEQ ID NO:77 (2D4H1-C3).

Exemplary HUI77 variants include, for example, the following antibodies:

-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:38; a heavy chain CDR2 referenced as SEQ ID NO:40; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (12F10Q).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:38; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (QH2b-A3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom1B6).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom1B8).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom1C3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:144; a heavy    chain CDR3 referenced as SEQ ID NO:103; a light chain CDR1    referenced as SEQ ID NO:149; a light chain CDR2 referenced as SEQ ID    NO:34; and a light chain CDR3 referenced as SEQ ID NO:36 (Qcom1D3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:151; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom1E3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:151; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom1H6).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:152; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:145 (Qcom1H7).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:148; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom2A4).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:115; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom2B11).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:40; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:153; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom2C1).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:116; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom2D9).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:116; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:36 (Qcom2E3).-   An antibody comprising a heavy chain CDR1 referenced as SEQ ID    NO:38; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain    CDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as    SEQ ID NO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a    light chain CDR3 referenced as SEQ ID NO:130 (Qh2b-B7).

The invention also provides grafted antibodies containing CDRs derivedfrom HUIV26 and HUI77, respectively. Such grafted CDRs include humanizedantibodies, in which CDRs from HUIV26 or HUI77 have been grafted or inwhich a CDR containing one or more amino acid substitutions is grafted.The CDRs can be grafted directly into a human framework, as disclosedherein. If desired, framework changes can also be incorporated bygenerating framework libraries. The optimization of CDRs and/orframework sequences can be performed independently and sequentiallycombined or can be performed simultaneously, as described in more detailbelow.

Thus, the invention additionally provides a grafted antibody in whichHUIV26 CDRs (SEQ ID NOS:20, 22, 24, 26, 28 and 30) are grafted into ahuman framework sequence. Also provided is a grafted antibody in whichHUI77 CDRs (SEQ ID NOS:32, 34, 36, 38, 40 and 42) are grafted into ahuman framework.

To generate grafted antibodies, donor CDRs of collagen-specificantibodies are grafted onto an antibody acceptor variable regionframework. Methods for grafting antibodies and generating CDR variantsto optimize activity have been described previously (WO 98/33919; WO00/78815; WO 01/27160). The procedure can be performed to achievegrafting of donor CDRs and affinity reacquisition in a simultaneousprocess. The methods similarly can be used, either alone or incombination with CDR grafting, to modify or optimize the bindingaffinity of a variable region. The methods for conferring donor CDRbinding affinity onto an acceptor variable region are applicable to bothheavy and light chain variable regions and as such can be used tosimultaneously graft and optimize the binding affinity of an antibodyvariable region.

The donor CDRs can be altered to contain a plurality of different aminoacid residue changes at all or selected positions within the donor CDRs.For example, random or biased incorporation of the twenty naturallyoccurring amino acid residues, or preselected subsets, can be introducedinto the donor CDRs to produce a diverse population of CDR species.Inclusion of CDR variant species into the diverse population of variableregions allows for the generation of variant species that exhibitoptimized binding affinity for a predetermined antigen.

A range of possible changes can be made in the donor CDR positions. Someor all of the possible changes that can be selected for change can beintroduced into the population of grafted donor CDRs. A single positionin a CDR can be selected to introduce changes or a variety of positionshaving altered amino acids can be combined and screened for activity.

One approach is to change all amino acid positions along a CDR byreplacement at each position with, for example, all twenty naturallyoccurring amino acids. The replacement of each position can occur in thecontext of other donor CDR amino acid positions so that a significantportion of the CDR maintains the authentic donor CDR sequence, andtherefore, the binding affinity of the donor CDR. For example, anacceptor variable region framework, either a native or alteredframework, can be grafted with a population of CDRs containing singleposition replacements at each position within the CDRs. Similarly, anacceptor variable region framework can be targeted for grafting with apopulation of CDRs containing more than one position changed toincorporate all twenty amino acid residues, or a subset of amino acids.One or more amino acid positions within a CDR, or within a group of CDRsto be grafted, can be altered and grafted into an acceptor variableregion framework to generate a population of grafted antibodies. It isunderstood that a CDR having one or more altered positions can becombined with one or more other CDRs having one or more alteredpositions, if desired.

A population of CDR variant species having one or more altered positionscan be combined with any or all of the CDRs which constitute the bindingpocket of a variable region. Therefore, an acceptor variable regionframework can be targeted for the simultaneous incorporation of donorCDR variant populations at one, two or all three recipient CDR locationsin a heavy or light chain. The choice of which CDR or the number of CDRsto target with amino acid position changes will depend on, for example,if a full CDR grafting into an acceptor is desired or whether the methodis being performed for optimization of binding affinity.

Another approach for selecting donor CDR amino acids to change forconferring donor CDR binding affinity onto an antibody acceptor variableregion framework is to select known or readily identifiable CDRpositions that are highly variable. For example, the variable regionCDR3 is generally highly variable. This region therefore can beselectively targeted for amino acid position changes during graftingprocedures to ensure binding affinity reacquisition or augmentation,either alone or together with relevant acceptor variable frameworkchanges, as described herein.

If desired, CDR variant populations having one or more altered aminoacid positions can be advantageously combined with a framework variantpopulation having one or more altered amino acid positions. Such acombination can result in beneficial combinations of changes, which areidentified by screening for an optimized activity.

The resultant population of CDR grafted variable regions thereforecontain a species corresponding to the authentic parent amino acidresidue at each position as well as a diverse number of differentspecies which correspond to the possible combinations and permutationsof the authentic parent amino acid residues together with the variantresidues at each of the selected CDR positions. Such a diversepopulation of CDR grafted variable regions are screened for an alteredvariable region species which retains donor CDR binding activity, orwhich has optimized binding activity.

An acceptor can be selected so that it is closely similar to thevariable region amino acid sequence harboring the donor CDRs. Inaddition, a variety of acceptors less closely related to the donorantibody can be used. Alternatively, a library of all possible orrelevant changes in the acceptor framework can be made and then screenedfor those variable regions, or heteromeric binding fragments thereof,that maintain or exhibit increased binding affinity compared to thedonor molecule. The donor CDRs can be grafted into a variety ofnaturally occurring acceptor frameworks or altered frameworks having oneor more changes or even a library containing changes at one or morepositions. Therefore, the applicability is not preconditioned on theavailability or search for an acceptor framework variable region similarto that of the donor.

The methods for conferring donor CDR binding affinity onto a variableregion can involve identifying the relevant amino acid positions in theacceptor framework that are known or predicted to influence a CDRconformation, or that are known or predicted to influence the spacialcontext of amino acid side chains within the CDR that participate inbinding, and then generating a population of altered variable regionspecies that incorporate a plurality of different amino acid residues atthose positions. For example, the different amino acid residues at thosepositions can be incorporated either randomly or with a predeterminedbias and can include all of the twenty naturally occurring amino acidresidues at each of the relevant positions. Subsets, including less thanall of the naturally occurring amino acids can additionally be chosenfor incorporation at the relevant framework positions. Including aplurality of different amino acid residues at each of the relevantframework positions ensures that there will be at least one specieswithin the population that will have framework changes which allows theCDRs to reacquire their donor binding affinity in the context of theacceptor framework variable region.

For humanizing an antibody, any of a variety of human frameworks can beselected for CDR grafting. For example, CDRs of HUIV26 or HUI77 can becloned into a variety of human framework sequences. The frameworks canbe generated using human germline genes encoding heavy and light chainvariable regions as well as J regions to obtain human frameworksequences for CDR grafting. Exemplary human framework nucleotidesequences include, for example, the framework sequences of DPK24 (VKIV)(SEQ ID NO:5), DP-54 (VHIII) (SEQ ID NO:7), DPK13 (VKII) (SEQ ID NO:13),DP-28 (VHII) (SEQ ID NO:15), as well as J regions JK1 (SEQ ID NO:217),JK2 (SEQ ID NO:218) and JH6 (SEQ ID NO:219). It is understood thatframework regions from any available germline sequence can be combinedwith any available J sequence, as desired, to generate a human frameworkfor grafting CDRs. For example, an alignment of mouse variable regionsof HUIV26 and HUI77 with an exemplary human framework is shown in FIGS.2C and 3C, respectively. A fusion of VKIV/JK2 light chain variableregion and VHIII/JH6 heavy chain variable region are aligned with HUIV26(FIG. 2C). A fusion of VKII/JK1 light chain variable region andVHIII/JH6 heavy chain variable region are aligned with HUI77 (FIG. 3C).An exemplary fusion of a germline and J region is shown in FIG. 3D,which is aligned with the HUI77 light chain. It is understood that anyavailable human framework can be selected for CDR grafting and, ifdesired, optimized by the methods disclosed herein. As disclosed herein,CDRs having beneficial mutations can be grafted into a variety offrameworks and have retained or improved activity (see Example III).

Selection of the relevant framework amino acid positions to alterdepends on a variety of criteria well known to those skilled it the art.One criteria for selecting relevant framework amino acids to change canbe the relative differences in amino acid framework residues between thedonor and acceptor molecules. Selection of relevant framework positionsto alter using this approach is simple and has the advantage of avoidingany subjective bias in residue determination or any bias in CDR bindingaffinity contribution by the residue.

Another criteria that can be used for determining the relevant aminoacid positions to change can be, for example, selection of frameworkresidues that are known to be important or to contribute to CDRconformation. For example, canonical framework residues are importantfor CDR conformation or structure. Targeting of a canonical frameworkresidue as a relevant position to change can identify a more compatibleamino acid residue in context with its associated donor CDR sequence.

The frequency of an amino acid residue at a particular frameworkposition is another criteria which can be used for selecting relevantframework amino acid positions to change. For example, comparison of theselected framework with other framework sequences within its subfamilycan reveal residues that occur at minor frequences at a particularposition or positions. Such positions harboring less abundant residuesare similarly applicable for selection as a position to alter in theacceptor variable region framework.

The relevant amino acid positions to change also can be selected, forexample, based on proximity to a CDR. In certain contexts, such residuescan participate in CDR conformation or antigen binding. Moreover, thiscriteria can similarly be used to prioritize relevant positions selectedby other criteria described herein. Therefore, differentiating betweenresidues proximal and distal to one or more CDRs is an efficient way toreduce the number of relevant positions to change.

Other criteria for selecting relevant amino acid framework positions toalter include, for example, residues that are known or predicted toreside in three-dimensional space near the antigen-CDR interface orpredicted to modulate CDR activity. Similarly, framework residues thatare known or predicted to form contacts between the heavy (V_(H)) andlight (V_(L)) chain variable region interface can be selected. Suchframework positions can affect the conformation or affinity of a CDR bymodulating the CDR binding pocket, antigen interaction or the V_(H) andV_(L) interaction. Therefore, selection of these amino acid positionsfor constructing a diverse population for screening of binding activitycan be used to identify framework changes which replace residues havingdetrimental effects on CDR conformation or compensate for detrimentaleffects of residues occurring elsewhere in the framework.

Other framework residues that can be selected for alteration includeamino acid positions that are inaccessible to solvent. Such residues aregenerally buried in the variable region and are therefore capable ofinfluencing the conformation of the CDR or V_(H) and V_(L) interactions.Solvent accessibility can be predicted, for example, from the relativehydrophobicity of the environment created by the amino acid side chainsof the polypeptide or by known three-dimensional structural data.

Following selection of relevant amino acid positions in the donor CDRs,as well as any relevant amino acid positions in the framework regionsdesired to be varied, amino acid changes at some or all of the selectedpositions can be incorporated into encoding nucleic acids for theacceptor variable region framework and donor CDRs. Altered framework orCDR sequences can be individually made and tested, or can besimultaneously combined and tested, if desired.

The variability at any or all of the altered positions can range from afew to a plurality of different amino acid residues, including alltwenty naturally occurring amino acids or functional equivalents andanalogues thereof.

Selection of the number and location of the amino acid positions to varyis flexible and can depend on the intended use and desired efficiencyfor identification of the altered variable region having a desirableactivity such as substantially the same or greater binding affinitycompared to the donor variable region. In this regard, the greater thenumber of changes that are incorporated into a altered variable regionpopulation, the more efficient it is to identify at least one speciesthat exhibits a desirable activity, for example, substantially the sameor greater binding affinity as the donor. Alternatively, where the userhas empirical or actual data to the affect that certain amino acidresidues or positions contribute disproportionally to binding affinity,then it can be desirable to produce a limited population of alteredvariable regions which focuses on changes within or around thoseidentified residues or positions.

For example, if CDR grafted variable regions are desired, a large,diverse population of altered variable regions can include all thenon-identical framework region positions between the donor and acceptorframework and all single CDR amino acid position changes. Alternatively,a population of intermediate diversity can include subsets, for example,of only the proximal non-identical framework positions to beincorporated together with all single CDR amino acid position changes.The diversity of the above populations can be further increased by, forexample, additionally including all pairwise CDR amino acid positionchanges. In contrast, populations focusing on predetermined residues orpositions which incorporate variant residues at as few as one frameworkand/or one CDR amino acid position can similarly be constructed forscreening and identification of an altered antibody variable region ofthe invention. As with the above populations, the diversity of suchfocused populations can be further increased by additionally expandingthe positions selected for change to include other relevant positions ineither or both of the framework and CDR regions. There are numerousother combinations ranging from few changes to many changes in either orboth of the framework regions and CDRs that can additionally beemployed, all of which will result in a population of altered variableregions that can be screened for the identification of at least one CDRgrafted altered variable region having desired activity, for example,binding activity to a cryptic collagen site. Those skilled in the artwill know, or can determine, which selected residue positions in theframework or donor CDRs, or subsets thereof, can be varied to produce apopulation for screening and identification of an altered antibody ofthe invention given the teachings and guidance provided herein.

Simultaneous incorporation of all of the CDR encoding nucleic acids andall of the selected amino acid position changes can be accomplished by avariety of methods known to those skilled in the art, including forexample, recombinant and chemical synthesis. For example, simultaneousincorporation can be accomplished by, for example, chemicallysynthesizing the nucleotide sequence for the acceptor variable region,fused together with the donor CDR encoding nucleic acids, andincorporating at the positions selected for harboring variable aminoacid residues a plurality of corresponding amino acid codons.

One such method well known in the art for rapidly and efficientlyproducing a large number of alterations in a known amino acid sequenceor for generating a diverse population of variable or random sequencesis known as codon-based synthesis or mutagenesis. This method is thesubject matter of U.S. Pat. Nos. 5,264,563 and 5,523,388 and is alsodescribed in Glaser et al. J. Immunology 149:3903 (1992). Briefly,coupling reactions for the randomization of, for example, all twentycodons which specify the amino acids of the genetic code are performedin separate reaction vessels and randomization for a particular codonposition occurs by mixing the products of each of the reaction vessels.Following mixing, the randomized reaction products corresponding tocodons encoding an equal mixture of all twenty amino acids are thendivided into separate reaction vessels for the synthesis of eachrandomized codon at the next position. For the synthesis of equalfrequencies of all twenty amino acids, up to two codons can besynthesized in each reaction vessel.

Variations to these synthesis methods also exist and include forexample, the synthesis of predetermined codons at desired positions andthe biased synthesis of a predetermined sequence at one or more codonpositions. Biased synthesis involves the use of two reaction vesselswhere the predetermined or parent codon is synthesized in one vessel andthe random codon sequence is synthesized in the second vessel. Thesecond vessel can be divided into multiple reaction vessels such as thatdescribed above for the synthesis of codons specifying totally randomamino acids at a particular position. Alternatively, a population ofdegenerate codons can be synthesized in the second reaction vessel suchas through the coupling of NNG/T nucleotides where N is a mixture of allfour nucleotides. Following synthesis of the predetermined and randomcodons, the reaction products in each of the two reaction vessels aremixed and then redivided into an additional two vessels for synthesis atthe next codon position.

A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the variant populations described herein.This modification is based on the two vessel method described above,which biases synthesis toward the parent sequence and allows the user toseparate the variants into populations containing a specified number ofcodon positions that have random codon changes.

Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

Following the above modification of codon-based synthesis, populationscontaining random codon changes at one, two, three and four positions aswell as others can be conveniently separated out and used based on theneed of the individual. Moreover, this synthesis scheme also allowsenrichment of the populations for the randomized sequences over theparent sequence since the vessel containing only the parent sequencesynthesis is similarly separated out from the random codon synthesis.

Other methods well known in the art for producing a large number ofalterations in a known amino acid sequence or for generating a diversepopulation of variable or random sequences include, for example,degenerate or partially degenerate oligonucleotide synthesis. Codonsspecifying equal mixtures of all four nucleotide monomers, representedas NNN, results in degenerate synthesis. Whereas partially degeneratesynthesis can be accomplished using, for example, the NNG/T codondescribed previously. Other methods well known in the art canalternatively be used such as the use of statistically predetermined, orvarigated, codon synthesis, which is the subject matter of U.S. Pat.Nos. 5,223,409 and 5,403,484.

Once the populations of altered variable region encoding nucleic acidshave been constructed as described above, they can be expressed togenerate a population of altered variable region polypeptides that canbe screened for binding affinity. For example, the altered variableregion encoding nucleic acids can be cloned into an appropriate vectorfor propagation, manipulation and expression. Such vectors are known orcan be constructed by those skilled in the art and should contain allexpression elements sufficient for the transcription, translation,regulation, and if desired, sorting and secretion of the alteredvariable region polypeptides. The vectors can be suitable for expressionin either procaryotic or eukaryotic host systems so long as theexpression and regulatory elements function in the respective hostsystem. The expression vectors can additionally include regulatoryelements for inducible or cell type-specific expression. One skilled inthe art will know which host systems are compatible with a particularvector and which regulatory or functional elements are sufficient toachieve expression of the polypeptides in soluble, secreted or cellsurface forms.

Appropriate host cells, include for example, bacteria and correspondingbacteriophage expression systems, yeast, avian, insect and mammaliancells. Methods for recombinant expression, screening and purification ofpopulations of altered variable regions or altered variable regionpolypeptides within such populations in various host systems are wellknown in the art and are described, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York (1992) and in Ausubel et al., Current Protocols in MolecularBiology, (Supplement 54), John Wiley & Sons, New York (2001). The choiceof a particular vector and host system for expression and screening ofaltered variable regions are known to those skilled in the art and willdepend on the preference of the user. Moreover, expression of diversepopulations of hetereomeric receptors in either soluble or cell surfaceform using filamentous bacteriophage vector/host systems is well knownin the art and is the subject matter of U.S. Pat. No. 5,871,974.

The expressed population of altered variable region polypeptides can bescreened for the identification of one or more altered variable regionspecies exhibiting optimized binding activity, for example, bindingaffinity substantially the same or greater than the donor CDR variableregion. Screening can be accomplished using various methods well knownin the art for determining the binding affinity of a polypeptide orcompound. Additionaly, methods based on determining the relativeaffinity of binding molecules to their partner by comparing the amountof binding between the altered variable region polypeptides and thedonor CDR variable region can similarly be used for the identificationof species exhibiting binding affinity substantially the same or greaterthan the donor CDR variable region. All of such methods can beperformed, for example, in solution or in solid phase. Moreover, variousformats of binding assays are well known in the art and include, forexample, immobilization to filters such as nylon or nitrocellulose;two-dimensional arrays, enzyme linked immunosorbant assay (ELISA),radioimmunoassay (RIA), panning and plasmon resonance. Such methods canbe found described in, for example, Harlow and Lane, supra, 1988.

For the screening of populations of polypeptides such as the alteredvariable region populations produced by the methods of the invention,immobilization of the populations of altered variable regions to filtersor other solid substrate can be advantageous because large numbers ofdifferent species can be efficiently screened for antigen binding. Suchfilter lifts allow for the identification of altered variable regionsthat exhibit substantially the same or greater binding affinity comparedto the donor CDR variable region. Alternatively, if the populations ofaltered variable regions are expressed on the surface of a cell orbacteriophage, panning on immobilized antigen can be used to efficientlyscreen for variants having antigen binding activity or to determine therelative binding affinity of species within the population.

Another affinity method for screening populations of altered variableregions polypeptides is a capture lift assay that is useful foridentifying a binding molecule having selective affinity for a ligand(Watkins et. al., (1997); WO 99/06834). This method employs theselective immobilization of altered variable regions to a solid supportand then screening of the selectively immobilized altered variableregions for selective binding interactions against the cognate antigenor binding partner. Selective immobilization functions to increase thesensitivity of the binding interaction being measured since initialimmobilization of a population of altered variable regions onto a solidsupport reduces non-specific binding interactions with irrelevantmolecules or contaminants which can be present in the reaction.

Another method for screening populations or for measuring the affinityof individual altered variable region polypeptides is through surfaceplasmon resonance (SPR). This method is based on the phenomenon whichoccurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (k_(on),) and disassociation rates (k_(off)).

Measurements of k_(on), and k_(off) values can be used identify alteredvariable regions or optimized variable regions that are therapeuticallymore efficacious. For example, an altered variable region, orheteromeric binding fragment thereof, can be more efficacious because ithas, for example, a higher k_(on), valued compared to variable regionsand heteromeric binding fragments that exhibit similar binding affinity.Increased efficacy is conferred because molecules with higher k_(on)values can specifically bind and inhibit their target at a faster rate.Similarly, a molecule of the invention can be more efficacious becauseit exhibits a lower k_(off) value compared to molecules having similarbinding affinity. Increased efficacy observed with molecules havinglower k_(off) rates can be observed because, once bound, the moleculesare slower to dissociate from their target. Although described withreference to the altered variable regions and optimized variable regionsof the invention, the methods described above for measuring associationand dissociation rates are applicable to essentially any antibody orfragment thereof for identifying more effective binders for therapeuticor diagnostic purposes.

Methods for measuring the affinity, including association anddissociation rates using surface plasmon resonance are well known in theart and can be found described in, for example, Jönsson and Malmquist,Advances in Biosensors, 2:291-336 (1992) and Wu et al. Proc. Natl. Acad.Sci. USA, 95:6037-6042 (1998). Moreover, one apparatus well known in theart for measuring binding interactions is a BIACORE 2000 instrumentwhich is commercially available through Pharmacia Biosensor, (Uppsala,Sweden).

Using any of the above described screening methods, as well as otherswell known in the art, an altered variable region having optimizedbinding activity, for example, binding affinity substantially the sameor greater than the donor CDR variable region is identified by detectingthe binding of at least one altered variable region within thepopulation to its antigen or cognate ligand. In addition to optimizingfor antigen binding activity, catalytic activity can also be included inan invention antibody and optimized using the methods disclosed hereinfor binding affinity optimization. Accordingly, the above methods can bemodified to include the addition of substrate and reactants to screenfor optimized catalytic activity. Comparison, either independently orsimultaneously in the same screen, with the donor variable region willidentify those binders that have substantially the same or greaterbinding affinity as the donor. Those skilled in the art will know, orcan determine using the donor variable region, binding conditions whichare sufficient to identify selective interactions over non-specificbinding.

Detection methods for identification of binding species within thepopulation of altered variable regions can be direct or indirect and caninclude, for example, the measurement of light emission, radioisotopes,calorimetric dyes and fluorochromes. Direct detection includes methodsthat function without intermediates or secondary measuring procedures toassess the amount of bound antigen or ligand. Such methods generallyemploy ligands that are themselves labeled with a detectable moiety, forexample, a radioactive, light emitting, fluorescent, calorimetric orenzyme moiety. In contrast, indirect detection includes methods thatfunction through an intermediate or secondary measuring procedure. Thesemethods generally employ molecules that specifically react with theantigen or ligand and can themselves be directly labeled with adetectable moiety or detected by a secondary reagent. For example, anantibody specific for a ligand can be detected using a secondaryantibody capable of interacting with the first antibody specific for theligand, again using the detection methods described above for directdetection. Moreover, for the specific example of screening for catalyticantibodies, the disappearance of a substrate or the appearance of aproduct can be used as an indirect measure of binding affinity orcatalytic activity.

Isolated variable regions exhibit binding affinity as single chains, inthe absence of assembly into a heteromeric structure with theirrespective V_(H) or V_(L) subunits. As such, populations of V_(H) andV_(L) altered variable regions polypeptides can be expressed alone andscreened for binding activity, for example, optimized activity havingsubstantially the same or greater binding affinity compared to the CDRdonor V_(H) or V_(L) variable region. Alternatively, populations ofV_(H) and V_(L) altered variable regions polypeptides can be coexpressedso that they self-assemble into heteromeric altered variable regionbinding fragments. The heteromeric binding fragment population can thenbe screened for species exhibiting binding affinity substantially thesame or greater than the CDR donor variable region binding fragment.

Employing the methods for simultaneously grafting and optimizing, or foroptimizing, it is possible to generate heteromeric variable regionbinding fragments having increases in affinities of greater than about2-fold, 3-fold, 4-fold, 5-fold, 8-fold or 10-fold. In particular,heteromeric variable region binding fragments can be generated havingincreases in affinities of greater than 12-fold, 15-fold, 20-fold, and25-fold as well as affinities greater than 50-fold, 100-fold, 200-fold,500-fold or 1000-fold compared to the donor or parent molecule.

Additionally, the methods described herein for optimizing are also areapplicable for producing catalytic heteromeric variable region fragmentsor for optimizing their catalytic activity. Catalytic activity can beoptimized by changing, for example, the on or off rate of substratebinding, the substrate binding affinity, the transition state bindingaffinity, the turnover rate (kcat) or the Km. Methods for measuringthese characteristics are well known in the art (see, for example Segel,Enzyme Kinetics, John Wiley & Sons, New York (1975)). Such methods canbe employed in the screening steps of the methods described above whenused for optimizing the catalytic activity of a heteromeric variableregion binding fragment.

Additionally, the methods for conferring donor CDR binding affinity ontoan antibody acceptor variable region framework are applicable forgrafting CDRs as described by Kabat et al., supra, Chothia et al., supraor MacCallum et al., supra. The methods similarly can be used forgrafting into an acceptor framework overlapping regions or combinationsof CDRs as described in Kabat et al., supra, Chothia et al., supra orMacCallum et al., supra. Generally, variable region CDRs are grafted byidentifying the boundries described by one of the CDR definitions knownin the art and set forth herein. However, because the methods aredirected to constructing and screening populations of CDR graftedaltered variable regions, which can incorporate relevant amino acidposition changes in both the framework and CDR regions, and suchvariations can, for example, compensate or augment amino acid changeselsewhere in the variable region, the exact boundry of a particular CDRor set of variable region CDRs can be varied. Therefore, the exact CDRregion to graft, whether it is the region described by Kabat et al.,Chothia et al. or MacCallum et al., or any combination thereof, willessentially depend on the preference of the user.

Similarly, the methods described previously for optimizing the bindingaffinity of an antibody also are applicable for use with essentially anyvariable region for which an encoding nucleic acid is, or can be made,available. As with the methods for conferring donor CDR bindingaffinity, many applications of the methods for optimizing bindingaffinity will be for modifying the binding affinity of CDR graftedvariable regions having human frameworks. Again, such molecules aresignificantly less antigenic in human patients and thereforetherapeutically valuable in the treatment of human diseases. However,the methods of the invention for optimizing the binding affinity of avariable region are applicable to all species of variable regions.Therefore, the invention includes binding affinity optimization ofvariable regions derived from human, mouse, rat, rabbit, goat andchicken, or any other desired species.

The methods of the invention have been described with reference tovariable regions and heteromeric variable region binding fragments.Those skilled in the art will understand that all of such methods areapplicable to whole antibodies and functional fragments thereof as wellas to regions and functional domains other than the antigen bindingvariable region of antibodies, if desired.

An association rate can be determined in any non-equilibrium mixtureincluding, for example, one formed by rapidly contacting a bindingpolypeptide and ligand or by rapidly changing temperature. Anon-equilibrium mixture can be a pre-equilibrium mixture. Apre-equilibrium mixture can be formed, for example, by contacting asoluble binding polypeptide and soluble ligand in a condition where theamount of total ligand and total binding polypeptide in the detectionchamber are constant. Measurements of association rates inpre-equilibrium mixtures can be made in formats providing rapid mixingof binding polypeptide with ligand and rapid detection of changingproperties of the binding polypeptide or ligand on a timescale ofmilliseconds or faster. Stopped flow and rapid quench flow instrumentssuch as those described below provide a convenient means to measurenon-equilibrium kinetics. The association rate can also be measured innon-equilibrium mixtures including, for example, solutions containinginsoluble species of binding polypeptide, ligand or binding polypeptidebound to ligand, or solutions containing variable concentrations oftotal ligand or total binding polypeptide. Measurement of an associationrate in a non-equilibrium mixture can be made in formats providingattachment of a ligand to a surface and continuous flow of a solutioncontaining the binding polypeptide over the surface, or vice-versa,combined with rapid detection of changing properties of the bindingpolypeptide, ligand or surface such that measurements are made on atimescale of milliseconds or faster. Examples of formats providingnon-equilibrium measurement of association rates include surface plasmonresonance instruments and evanescent wave instruments.

Association rate measurements can be made by detecting the change in aproperty of the binding polypeptide or ligand that exists between thebound and unbound state or by detecting a change in the surroundingenvironment when binding polypeptide and ligand associate. Properties ofthe binding polypeptide or ligand that can change upon association andthat can be used to measure association rates include, for example,absorption and emission of heat, absorption and emission ofelectromagnetic radiation, affinity for a receptor, molecular weight,density, mass, electric charge, conductivity, magnetic moment of nuclei,spin state of electrons, polarity, molecular shape, or molecular size.Properties of the surrounding environment that can change when bindingpolypeptide associates with ligand include, for example, temperature andrefractive index of surrounding solvent.

Formats for measuring association rates in pre-equilibrium mixturesinclude, for example, stopped flow kinetic instruments and rapid quenchflow instruments. A stopped flow instrument can be used to pushsolutions containing a binding polypeptide and ligand from separatereservoirs into a mixing chamber just prior to passage into a detectioncell. The instrument can then detect a change in one or more of theabove described properties to monitor progress of the binding event. Arapid quench flow instrument can be used to rapidly mix a solutioncontaining a binding polypeptide with a solution containing a ligandfollowed by quenching the binding reaction after a finite amount oftime. A change in one or more of the above described properties can thenbe detected for quenched mixtures produced by quenching at differenttimes following mixing. Quenching can be performed for example byfreezing or addition of a chemical quenching agent so long as thequenching step does not inhibit detection of the property relied uponfor measurement of binding rate. Thus, a rapid quench instrument can beuseful, for example, in situations where spectroscopic detection is notconvenient. A variety of instruments are commercially available fromvendors such as KinTek Corp. (State College, Pa.) and Hi-Tech Scientific(Salisbury, UK).

Formats for measuring association rates in non-equilibrium mixturesinclude, for example, surface plasmon resonance and evanescent waveinstruments. Surface plasmon resonance and evanescent wave technologyutilize a ligand or binding polypeptide attached to a biosensor surfaceand a solution containing either the binding polypeptide or ligandrespectively that is passed over the biosensor surface. The change inrefractive index of the solution that occurs at the surface of a chipwhen binding polypeptide associates with ligand can be measured in atime dependent fashion. For example, surface plasmon resonance is basedon the phenomenon which occurs when surface plasmon waves are excited ata metal/liquid interface. Light is directed at, and reflected from, theside of the surface not in contact with sample, and SPR causes areduction in the reflected light intensity at a specific combination ofangle and wavelength. Biomolecular binding events cause changes in therefractive index at the surface layer, which are detected as changes inthe SPR signal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (k_(on)) and disassociation rates (k_(off)). Surfaceplasmon resonance instruments are available in the art including, forexample, the BIACORE instrument, IBIS system, SPR-CELLIA system,SPREETA, and Plasmon SPR and evanescent wave technology is available inthe Iasys system as described, for example, in Rich and Myszka, Curr.Opin. Biotech. 11:54-61 (2000).

Another method for measuring binding affinity includes comparativeELISA. As disclosed herein, an approximation of changes in affinitybased on shifts in half-maximal binding was used to identify k_(on) andk_(off) values relative to wild type (Example III). Such a method isparticularly useful for screening large numbers of variants, whereas theabove-described methods can be used for detailed analysis of bindingactivity.

The association rate can be determined by measuring a change in aproperty of a ligand or binding polypeptide at one or more discreet timeintervals during the binding event using, for example, the methodsdescribed above. Measurements determined at discreet time intervalsduring the binding event can be used to determine a quantitative measureof association rate or a relative measure of association rate.Quantitative measures of association rate can include, for example, anassociation rate value or k_(on) value. Quantitative values ofassociation rate or k_(on) can be determined from a mathematical orgraphical analysis of a time dependent measurement. Such analyses arewell known in the art and include algorithms for fitting data to a sumof exponential or linear terms or algorithms for computer simulation tofit data to a binding model as described for example in Johnson, Cur.Opin. Biotech. 9:87-89 (1998), which is incorporated herein byreference.

Association rates can be determined from mixtures containing insolublespecies or variable concentrations of total ligand or total bindingpolypeptide using mathematical and graphical analyses such as thosedescribed above if effects of mass transport are accounted for in thereaction. One skilled in the art can account for mass transport bycomparing association rates under conditions having similar limitationswith respect to mass transport or by adjusting the calculatedassociation rate according to models available in the art including, forexample those described in Myszka et al., Biophys. J. 75:583-594 (1998),which is incorporated herein by reference.

A higher value of either the association rate or k_(on) is generallyindicative of improved therapeutic potency. Thus, quantitativedeterminations provide an advantage by allowing comparison between anassociation rate of a binding polypeptide and a therapeutic controldetermined by different methods so long as the methods used areunderstood by one skilled in the art to yield consistent results.

A relative measure of association rate can include, for example,comparison of association rate for two or more binding polypeptidesbinding to ligand under similar conditions or comparison of associationrate for a binding polypeptide binding to ligand with a predefined rate.Comparison of association rate for two or more binding polypeptides caninclude a standard of known association rate or a molecule of knowntherapeutic effect. A predefined rate used for comparison can bedetermined by calibrating the measurement relative to a previouslymeasured rate including, for example, one available in the scientificliterature or in a database. An example of a comparison with apredefined rate is selection of the species of binding polypeptide boundto ligand at a discreet time interval defined by the predefined rate byusing a time actuated selection device.

For purposes of comparison, the association rate of a bindingpolypeptide and ligand can be determined relative to association ratefor a therapeutic control and the same ligand. A comparison can also bemade according to a quantitative association rate for bindingpolypeptide and ligand compared to a quantitative association rate for atherapeutic control and ligand. Relative or quantitative associationrates can be determined by the methods described above. Determination ofassociation rates for a binding polypeptide associating with a ligandcan be performed simultaneously with a binding polypeptide andtherapeutic control or at separate times, provided conditions aresufficiently similar in each assay to allow valid comparison. Thus,association rate determined for a binding polypeptide can be compared toa previously measured association rate for a therapeutic control.

A binding polypeptide having improved therapeutic potency can bedistinguished from a binding polypeptide that has an increased K_(a) fora ligand but not improved therapeutic potency. Methods for identifying atherapeutic binding polypeptide based on K_(a) rely on an equilibriummeasurement which, absent time dependent measurements made in anon-equilibrium condition, are inaccurate for identifying a bindingpolypeptide having increased association rate and therefore improvedtherapeutic potency. According to the relationship K_(a)=k_(on)/k_(off),an increased K_(a) for association of a binding polypeptide and ligandcan be due to changes in k_(on) or k_(off). For example, a bindingpolypeptide having improved therapeutic potency can have a reduced K_(a)if a reduction in k_(off) occurs that over compensates for an increasein k_(on). Thus, changes in K_(a), being influenced by changes ink_(off), do not unambiguously correlate with changes in therapeuticpotency since binding polypeptides having improved therapeutic potencycan display either reduced or increased K_(a).

For optimization of binding activity of an antibody of the invention,the fold increase in association rate can be indicated by an increase ink_(on). Therefore, k_(on) can be about 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more using methodsdescribed herein. The k_(on) can be at least about 1×10² M⁻¹s⁻¹, 2×10²M⁻¹s⁻¹, 5×10² M⁻¹s⁻¹, 1×10³ M⁻¹s⁻¹, 2×10³ M⁻¹s⁻¹, 5×10³ M⁻¹s⁻¹, 1×10⁴M⁻¹s⁻¹, 2×10⁴ M⁻¹s⁻¹, 5×10⁴ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹, 2×10⁵ M⁻¹s⁻¹, or 3×10⁵M⁻¹s⁻¹. The k_(on) can also be increased to at least about 5×10⁵ M⁻¹s⁻¹,7×10⁵ M⁻¹s⁻¹, 9×10 M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹, 3×10⁶M⁻¹s⁻¹, 5×10⁶ M⁻¹s⁻¹,7×10⁶ M⁻¹s⁻¹, 9×10⁶ M⁻¹s⁻¹ or 1×10⁷ M⁻¹s⁻¹ or more. Furthermore, theincrease in k_(on) resulting in improved therapeutic potency can beindependent of an effect of a change in K_(a) for the bindingpolypeptide. The binding polypeptide having an increase in k_(on) canhave a K_(a) value similar to K_(a) for its parent polypeptide or aK_(a) value lower than K_(a) for its parent polypeptide.

The invention also provides nucleic acids encoding the antibodies andCDRs of the invention. The invention further provides nucleic acidsencoding the mouse antibodies HUIV26 (SEQ ID NOS:1 and 3) and HUI77 (SEQID NOS:5 and 7) (see FIGS. 2 and 3). Further provided are nucleic acidsencoding HUIV26 CDRs (SEQ ID NOS:20, 22, 24, 26, 28 and 30) and encodingHUI77 CDRs (SEQ ID NOS:32, 34, 36, 38, 40 and 42). Such nucleic acidsinclude nucleic acids having degenerate codons encoding any or all ofthe amino acids in the CDRs. For example, the invention provides nucleicacids encoding HUIV26 CDRs: V_(L) CDR1, SEQ ID NOS:19; V_(L) CDR2, SEQID NO:21; V_(L) CDR3, SEQ ID NO:23; V_(H) CDR1, SEQ ID NO:25; V_(H)CDR2, SEQ ID NO:27; and V_(H) CDR3, SEQ ID NO:29. The invention alsoprovides nucleic acids encoding HUI77 CDRs: V_(L) CDR1, SEQ ID NOS:31;V_(L) CDR2, SEQ ID NO:33; V_(L) CDR3, SEQ ID NO:35; V_(H) CDR1, SEQ IDNO:37; V_(H) CDR2, SEQ ID NO:39; and V_(H) CDR3, SEQ ID NO:41. Alsoincluded are degenerate versions of such nucleic acids such that theyencode the amino acid sequences referenced as SEQ ID NOS:20, 22, 24, 26,28 and 30 for HUIV26 and SEQ ID NOS:32, 34, 36, 38, 40 and 42 for HUI77.

Further provided are nucleic acids encoding a HUIV26 or HUI77 CDRcontaining one or more amino acid subsitutions. For example, theinvention provides nucleic acids encoding the CDRs of HUIV26 and HUI77having single or multiple amino acid substitutions, as disclosed herein.If a nucleic acid encoding a CDR having one or more amino acidsubstitution is derived, for example, from one of SEQ ID NOS:19, 21, 23,25, 27 or 29 for HUIV26 or SEQ ID NOS:31, 33, 35, 37, 39 or 41 forHUI77, the amino acid substitutions can be encoded by any of thecorresponding degenerate codons for that amino acid. Nucleic acidsencoding such CDR variants can also include degenerate codons at any orall of the wild type amino acid positions.

Throughout the application, various nucleic acids and oligonucleotideprimers, in addition to the naturally occurring nucleotides A, C, G, Tor U, refer to standard abbreviations: R=G or A; Y=T/U or C; M=A or C;K=G or T/U; S=G or C; W=A or T/U; B=G, C or T/U; D=A, G or T/U; H=A, Cor T/U; V=A, G or C; N=any nucleotide.

The antibodies of the invention have binding activity for a crypticcollagen epitope. The HUIV26 and HUI77 antibodies have been shown totarget to angiogenic vasculature (see Xu et al., supra, 2001; WO00/40597). Accordingly, the grafted HUIV26 and HUI77 antibodies of theinvention, which specifically bind to a cryptic collagen epitope,similarly can target to angiogenic vasculature. One of the mostsignificant and important aspects of the monoclonal antibodies HUIV26and HUI77, and the grafted forms thereof disclosed herein, is that oftheir specificity. It is expected that systemic administration ofantibodies of the invention will have minimal if any toxic side effectssince the cryptic epitope(s) that is recognized by the HUIV26 and HUI77antibodies is/are not exposed in mature native triple helical collagenbut is only exposed upon denaturaion, for example, heat denaturation orproteolytic denaturation. Thus, little, if any, binding under normalphysiological conditions is expected.

Moreover, the cryptic collagen domain(s) to which HUIV26 and HUI77 bindrepresents a novel therapeutic target for the treatment of numerousneovascular diseases including tumor growth and metastasis, diabeticretinopathy and other related ocular diseases such as maculardegeneration, psoriasis, and rheumatoid arthritis. Other exemplarydiseases associated with angiogenesis include, but are not limited to,inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi's sarcoma and the likecancers which require neovascularization to support tumor growth. Otherexemplary tumors include melanoma, carcinoma, sarcoma, fibrosarcoma,glioma and astrocytoma, and the like.

Thus, the methods of the invention can be used to treat an individualhaving a disease associated with angiogenesis, including those describedabove. The methods can be used to ameliorate a sign or symptomassociated with a disease. For example, in the case of cancer treatment,the methods can be used to inhibit tumor growth. One skilled in the artwill know or can readily determine an appropriate sign or symptomassociated with a disease suitable for determining the effectiveness ofa therapeutic application using an antibody of the invention.

The antibodies of the invention can also be used as an importantdiagnostic and imaging reagent for the early detection of aberrantneovascularization associated with invasive tumor growth and metastasis.The antibodies of the invention can also be used in staging and gradingof tumors since invasive tumor in contrast to benign lesions are likelyto be associated with degradation of the surrounding basement membrane.

Thus, the invention provides a method of targeting angiogenicvasculature, comprising administering an antibody, or functionalfragment thereof, the antibody or functional fragment thereof havingspecific binding activity for a cryptic collagen epitope, wherein theantibody or functional fragment is an antibody of the invention. Forexample, the antibodies can comprise one or more CDRs, including wildtype CDRs or variants thereof, of the HUIV26 and HUI77 antibodies, asdisclosed herein. The methods of targeting angiogenic vasculature can beused for therapeutic and/or diagnostic purposes.

For therapeutic purposes, the antibody, or functional fragment thereof,can be administered as a therapeutic agent itself or can furthercomprise a therapeutic moiety. In the case of a therapeutic moiety, themoiety can be a drug such as a chemotherapeutic agent, cytotoxic agent,toxin, or anti-angiogenic agent, which refers to a molecule that reducesor inhibits angiogenesis. For example, a cytotoxic agent can be aradionuclide or chemical compound. Exemplary radionuclides useful astherapeutic agents include, for example, X-ray or y-ray emitters. Inaddition, a moiety can be a drug delivery vehicle such as a chamberedmicrodevice, a cell, a liposome or a virus, which can contain an agentsuch as a drug or a nucleic acid.

Exemplary therapeutic agents include, for example, the anthracyclin,doxorubicin, which has been linked to antibodies and theantibody/doxorubicin conjugates have been therapeutically effective intreating tumors (Sivam et al., Cancer Res. 55:2352-2356 (1995); Lau etal., Bioorg. Med. Chem. 3:1299-1304 (1995); Shih et al., Cancer Immunol.Immunother. 38:92-98 (1994)). Similarly, other anthracyclins, includingidarubicin and daunorubicin, have been chemically conjugated toantibodies, which have delivered effective doses of the agents to tumors(Rowland et al., Cancer Immunol. Immunother. 37:195-202 (1993);Aboud-Pirak et al., Biochem. Pharmacol. 38:641-648 (1989)).

In addition to the anthracyclins, alkylating agents such as melphalanand chlorambucil have been linked to antibodies to producetherapeutically effective conjugates (Rowland et al., Cancer Immunol.Immunother. 37:195-202 (1993); Smyth et al., Immunol. Cell Biol.65:315-321 (1987)), as have vinca alkaloids such as vindesine andvinblastine (Aboud-Pirak et al., supra, 1989; Starling et al., Bioconj.Chem. 3:315-322 (1992)). Similarly, conjugates of antibodies andantimetabolites such as 5-fluorouracil, 5-fluorouridine and derivativesthereof have been effective in treating tumors (Krauer et al., CancerRes. 52:132-137 (1992); Henn et al., J. Med. Chem. 36:1570-1579 (1993)).Other chemotherapeutic agents, including cis-platinum (Schechter et al.,Int. J. Cancer 48:167-172 (1991)), methotrexate (Shawler et al., J.Biol. Resp. Mod. 7:608-618 (1988); Fitzpatrick and Garnett, AnticancerDrug Des. 10:11-24 (1995)) and mitomycin-C (Dillman et al., Mol.Biother. 1:250-255 (1989)) also are therapeutically effective whenadministered as conjugates with various different antibodies. Atherapeutic agent can also be a toxin such as ricin.

A therapeutic agent can also be a physical, chemical or biologicalmaterial such as a liposome, microcapsule, micropump or other chamberedmicrodevice, which can be used, for example, as a drug delivery system.Generally, such microdevices, should be nontoxic and, if desired,biodegradable. Various moieties, including microcapsules, which cancontain an agent, and methods for linking a moiety, including achambered microdevice, to an antibody of the invention are well known inthe art and commercially available (see, for example, “Remington'sPharmaceutical Sciences” 18th ed. (Mack Publishing Co. 1990), chapters89-91; Harlow and Lane, Antibodies: A laboratory manual (Cold SpringHarbor Laboratory Press 1988)).

For diagnostic purposes the antibody, or functional fragment thereof,can further comprise a detectable moiety. A detectable moiety can be,for example, a radionuclide, fluorescent, magnetic, calorimetric moeity,and the like. For in vivo diagnostic purposes, a moiety such as a gammaray emitting radionuclide, for example, indium-111 or technitium-99, canbe linked to an antibody of the invention and, following administrationto a subject, can be detected using a solid scintillation detector.Similarly, a positron emitting radionuclide such as carbon-11 or aparamagnetic spin label such as carbon-13 can be linked to the moleculeand, following administration to a subject, the localization of themoiety can be detected using positron emission transaxial tomography ormagnetic resonance imaging, respectively. Such methods can identify aprimary tumor as well as a metastatic lesion.

For diagnostic purposes, the antibodies of the invention can be used todetermine the levels of denatured collagen in a tissue or in a bodilyfluid. The level of denatured collagen can be determined in a tissuesample obtained from an individual, for example, by tissue biopsy.Exemplary bodily fluids include, but are not limited to, serum, plasma,urine, synovial fluid, and the like.

The invention also provides a method of inhibiting angiogenesis byadministering an antibody, or functional fragment thereof, where theantibody or functional fragment thereof has specific binding activityfor a cryptic collagen epitope, where the antibody comprises one or moreCDRs of the invention. For example, an antibody of the invention can beadministered so that angiogenesis is inhibited in a tissue of anindividual. The invention further provides a method of targeting a tumorby administering an invention antibody. The invention also provides amethod of inhibiting tumor growth by administering an antibody, orfunctional fragment thereof, of the invention.

The antibodies of the invention can also be used for in vivo or in vitrodiagnostic applications. Thus, the invention provides a method ofdetecting angiogenic vasculature by contacting angiogenic vasculaturewith an antibody, or functional fragment thereof, of the invention.Angiogenic vasculature can be imaged in vivo by administering anantibody of the invention, either alone or attached to a detectablemoiety, to an individual. The angiogenic vasculature can thus bedetected in vivo. Alternatively, the antibody can be administered to atissue obtained from an individual, for example, a tissue biopsy, suchthat an antibody of the invention can be used in vitro for diagnosticpurposes to detect angiogenic vasculature.

A therapeutic or detectable moiety can be coupled to an antibody of theinvention, or functional fragment thereof, by any of a number of wellknown methods for coupling or conjugating moieties. It is understoodthat such coupling methods allow the attachment of a therapeutic ordetectable moiety without interfering or inhibiting the binding activityof the antibody, that is, the ability to bind a cryptic collagen site.Methods for conjugating moieties to an antibody of the invention, orfunctional fragment thereof, are well known to those skilled in the art(see, for example, Hermanson, Bioconjugate Techniques, Academic Press,San Diego (1996)).

When administered to a subject, the antibody of the invention isadministered as a pharmaceutical composition containing, for example,the antibody and a pharmaceutically acceptable carrier. As disclosedherein, the antibody can be coupled to a therapeutic or detectablemoiety. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the conjugate. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art will know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition. The pharmaceutical composition alsocan contain an agent such as a cancer therapeutic agent.

One skilled in the art will know that a pharmaceutical compositioncontaining an antibody of the invention can be administered to a subjectby various routes including, for example, orally or parenterally, suchas intravenously. The composition can be administered by injection or byintubation. The pharmaceutical composition also can be an antibodylinked to liposomes or other polymer matrices, which can haveincorporated therein, for example, a drug such as a chemotherapeuticagent (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRCPress, Boca Raton Fla. (1993), which is incorporated herein byreference). Liposomes, for example, which consist of phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

For diagnostic or therapeutic methods disclosed herein, an effectiveamount of the antibody and therapeutic moiety is administered to thesubject. As used herein, the term “effective amount” means the amount ofthe pharmaceutical composition that produces the desired effect. Aneffective amount often will depend on whether the antibody itself isadministered or whether the antibody is linked to a moiety and the typeof moiety. Thus, a lesser amount of a radiolabeled molecule can berequired for imaging as compared to the amount of a radioactivedrug/antibody conjugate administered for therapeutic purposes. Aneffective amount of a particular antibody/moiety for a specific purposecan be determined using methods well known to those in the art. Oneskilled in the art can readily determine an appropriate dose of anantibody of the invention for an effective amount for therapeutic ordiagnostic purposes.

For therapeutic or in vivo diagnostic purposes, it is understood thatany of a variety of methods of administration can be used so long as theadministration is effective for a desired purpose. Such methods ofadministration include, for example, intravenous, transdermal,intrasynovial, intramuscular, intratumoral, intraocular, intranasal,intrathecal, topical, oral, or the like. One skilled in the art canreadily determine an appropriate mode of administration depending on thedesired therapeutic effect or desired diagnostic purpose.

Furthermore, it is understood that for therapeutic or diagnosticapplications, an antibody of the invention in general is administered toa mammal, for example, a human. Applications of an antibody of theinvention for domestic animals or agricultural purposes include othermammals, for example, a non-human primate, pig, cow, horse, goat, sheep,mule, donkey, dog, cat, rabbit, mouse, rat, and the like.

It is understood that any of the therapeutic methods disclosed hereinusing an antibody of the invention can be used in combination with othertherapeutic methods. For example, an antibody of the invention, eitherthe antibody itself or an antibody attached to a therapeutic agent, canbe administered simultaneously or sequentially with other therapeutictreatment regimens. For example, an antibody of the invention can beadministered alone or in combination with another therapeutic treatment,including any of the therapeutic drugs disclosed herein as well as otherdrugs well known to those skilled in the art for treating a particulardisease. For example, in the case of treating a cancer, an antibody ofthe invention can be administered simultaneously or sequentially withanother chemotherapeutic agent such as a drug or radionuclide.Similarly, an antibody of the invention can be combined with othertreatment regimens such as surgery by administering the antibody before,during or after surgery. One skilled in the art will know or can readilydetermine a desirable therapeutic treatment to be used in combinationwith an antibody of the invention, as desired. Thus, an antibody of theinvention can be administered in conjunction with other therapeuticregimens, including but not limited to chemotherapy, radiation therapy,surgery, and the like.

The invention additionally provides a method of inhibiting metastasisusing an antibody of the invention. The method can include the step ofadministering an antibody, or functional fragment thereof, havingbinding activity for a cryptic collagen epitope. The antibody can be,for example, an antibody comprising one or more CDRs having a least oneamino acid substitution in one or more heavy or light chain CDRs ofantibodies HUIV26 and HUI77. As used herein, inhibiting metastasisrefers to decreasing the number and/or size of metastatic sites remotefrom a primary tumor site. The method of inhibiting metastasis caninvolve using an antibody of the invention that blocks adhesion of tumorcells to a cryptic collagen epitope that is exposed after remodeling oftissues by the action of collagen-degrading enzymes secreted by tumorcells.

As disclosed herein, a variant of HUI77 having one or more amino acidsubstitutions in one or more CDRs inhibited proliferation of melanomacells in vitro (see Example VI). An antibody of the invention can blockaccess to or inhibit binding of a survival or proliferative signaldelivered to a tumor cell. Thus, the invention also provides a method oftargeting a tumor cell by administration of an antibody of the inventionhaving binding activity for a cryptic collagen epitope that blocksaccess to a survival or proliferative signal delivered to the tumor cellby a cryptic collagen site.

For methods of inhibiting angiogenesis, the angiogenic vasculature canbe associated with a tumor. The methods of the invention can also beused to inhibit tumor growth directly, alone or in combination withinhibiting angiogenic vasculature of the tumor. The methods of theinvention can additionally be used to inhibit metastasis, alone or incombination with inhibiting tumor angiogenic vasculature and/or tumorgrowth. Exemplary tumors include, but are not limited to, thosedisclosed herein, including melanoma, carcinoma, sarcoma, fibrosacroma,glioma, astrocytoma, and the like. Methods for testing the effect aHUIV26 or HUI77 variant for inhibition of angiogenesis or inhibition oftumor growth can be performed as described previously using, forexample, assays such as the rat corneal micropocket angiogenesis assay,chick embryo tumor growth assay, or SCID mouse tumor growth assay, asdescribed in Xu et al., supra, 2001, or any other well known assays formeasuring inhibition of angiogenesis, inhibition of tumor growth, orinhibition of metastasis.

The methods of the invention can also be applied to inhibiting non-tumorangiogenic vasculature. Such applications to non-tumor angiogenicvasculature can include tissue that is inflamed and in whichangiogenesis is occurring. Exemplary non-tumor diseases associated withangiogenic vasculature suitable for treatment with an antibody of theinvention include, but are not limited to, those disclosed herein,including arthritis, ocular disease, retinal disease, hemangioma, andthe like. The antibodies of the invention can also be used to inhibitpsoriasis, macular degeneration, restenosis, and the like, or any tumoror non-tumor disease associated with increased accessibility of acryptic collagen epitope for which an antibody of the invention hasbinding activity.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cloning of Heavy and Light Chain Variable Regions of HUIV26and HUI77 Antibodies

This example describes the cloning of HUIV26 and HUI77 antibody variableregions.

The variable regions of the HUIV26 and HUI77 antibodies were cloned fromhybridomas expressing these mouse monoclonal antibodies and sequenced.Briefly, total mRNA was isolated from the respective mouse hybridomacells using Oligotex® Direct mRNA Micro kit (Qiagen; Valencia Calif.).First strand cDNA was synthesized from the mRNA using SUPERSCRIPTPreamplification System (GibcoBRL/Invitrogen; Carlsbad Calif.). Antibodyvariable region sequences were amplified by PCR using a set of 5′primers designed for signal sequences of mouse light chains or heavychains to pair with single 3′ primer to mouse kappa chain constantregion for V_(L) or IgM CH1 region for V_(H) sequences. The sequences ofthe 5′ primers for the signal peptide of mouse antibody heavy and lightchain as well as constant region primers are shown in FIG. 1. The 3′primer for mouse kappa light chain constant region (primer 2650; SEQ IDNO:212) corresponds to amino acids 115-123. The 3′ primer for mouse IgMCH1 region (primer 2656; SEQ ID NO:213) corresponds to amino acids121-114. The 3′ primer for mouse IgM CH1 region (primer 2706; SEQ IDNO:214) corresponds to amino acids 131-124.

The DNA fragments were isolated from PCR reactions, with a main productof about 400 bp in length. The DNA fragments were cloned into the pCR2.1vector. The inserted DNA fragments were sequenced with both forward andreversed M13 primers. The DNA sequences were compared with an antibodysequence database. The N-terminal amino acid sequence of the HUIV26 andHUI77 antibodies were determined, and the sequences of the DNA fragmentswere also compared to the N-terminal amino acid sequences of thecorresponding antibody.

The HUIV26 V_(L) encoding nucleic acid was cloned with 5′ primer mK2(primer 2664; SEQ ID NO:185) and 3′ primer 2650 (SES ID NO:212). Apartial sequence of HUIV25 V_(L) is ATCTTCTTGCTGTTCTGGGTATCTGGAACCTGTGGG(SEQ ID NO:215), with the MK2 primer underlined and the partial sequencecoding for mouse signal peptide in italics. The HUIV26 V_(H) encodingnucleic acid was cloned with 5′ primer MH12 (primer 2731; SEQ ID NO:203)and 3′ primer 2706 (SEQ ID NO:214).

The HUI77 V_(L) encoding nucleic acid was cloned with 5′ primer mK1(primer 2663; SEQ ID NO:184) and 3′ primer 2650 (SEQ ID NO:212). Apartial sequence of HUI77 V_(L) is TTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT(SEQ ID NO:216), with the mK1 primer underlined and the partial sequencecoding for mouse signal peptide in italics. The HUI77 encoding nucleicacid was cloned with 5′ primers MH15 (primer 2734; SEQ ID NO:206) orMH16 (primer 2735; SEQ ID NO:207) and 3′ primer 2656 (SEQ ID NO:213).

The sequences of the heavy and light chain nucleotide and amino acidsequences for HUIV26 and HUI77 are shown in FIGS. 2 and 3, respectively.Using the numbering system of Kabat, supra, the CDRs of the heavy andlight chains were identified for each of the HUIV26 and HUI77 antibodies(underlined in FIGS. 2C and 3C).

An alignment of the HUI77 V_(L) nucleotide sequence (SEQ ID NO:9) withthe nucleotide sequence of the human framework fusion DPK13/JK1 (SEQ IDNO:17) is shown in FIG. 3D. The corresponding light chain amino acidsequences are referenced as SEQ ID NO:10 and SEQ ID NO:18 for HUI77 andDPK13/JK1, respectively.

This example describes the cloning and the sequence of mouse antibodiesHUIV26 and HUI77.

EXAMPLE II Generation of CDR Variant Libraries of HUIV26 and HUI77Antibodies

This example describes the generation of CDR variant libraries of HUIV26and HUI77 antibodies for CDR optimization.

The CDR3 regions of antibodies HUIV26 and HUI77 were optimized bygenerating a library of CDR variants. Primers for light chain CDR3 andheavy chain CDR3 were used to generate a library of CDR3 variants, wherethe primer was synthesized to encode more than one amino acid one ormore positions in CDR3. Following synthesis of primers encoding CDR3variants, the variant CDR3 regions were assembled into light chain(V_(L)) and heavy chain (V_(H)) regions.

Briefly, humanized V_(L) and V_(H) genes of HUI77 and HUIV26 antibodieswere assembled with the primers shown in FIGS. 4A and 5A, respectively,using PCR or primer-elongation-ligantion. Variable region genescontaining CDR3 mutations were assembled by replacing the wild type CDR3primer (IV26-17, IV26-h7, I77-17 or I77-h7) with the group of mutantprimers corresponding to that CDR. The assembled variable regions werethen amplified and asymmetrically biotinylated on plus strand by PCRusing primers B-pelB and 224 for V_(L) and B-phA and 1200a for H_(V)genes. The primers for amplification of humanized V_(L) and V_(H)sequences and the isolation of minus strand DNA were: B-pelB, Biotin-TTACTC GCT GCC CAA CCA GCC ATG GCC (SEQ ID NO:220); 224, GAC AGA TGG TGCAGC CAC AGT (SEQ ID NO:221); B-phoA, Biotin-TTA CTG TTT ACC CCT GTG ACAAAA GCC (SEQ ID NO:222); and 1200a, GAA GAC CGA TGG GCC CTT GGT (SEQ IDNO:223).

The assembled V_(L) and V_(H) regions were introduced into a Fabexpression vector by mutagenesis. Briefly, the non-biotinylated minusstrands were isolated after binding the PCR products toNEUTRAVIDIN-conjugated magnetic beads and introduced into the Fabexpression vector IX-104CSA by hybridization mutagenesis (Kristensson etal., Vaccines 95, pp. 39-43, Cold Spring Harbor Laboratory, Cold SpringHarbor (1995); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Wuet al., J. Mol. Bio. 294:151-162 (1999)).

Three humanization-CDR3-mutation libraries were constructed for each theHUI77 and HUIV26 antibodies. The three libraries introduced randommutations but differed in CDR3 mutations. One library had mutations onlyin LCDR3, the second library had mutations only in HCDR3, and the thirdlibrary had mutations in both LCDR3 and HCDR3.

Methods essentially the same as those described above for CDR3mutagenesis were also performed on CDR1 and CDR2 of the HUIV26 and HUI77antibodies. After assembling into a Fab expression vector, the Fabscontaining HUIV26 and HUI77 variant CDRs were expressed in bacteria andtested for binding to denatured collagen. The mutant libraries werescreened with filter lift screening and ELISA. The assays were performedessentially as described previously (Huse et al., J. Immunol.149:3914-3920 (1992); Watkins et al., Anal. Biochem. 253:37-45 (1997)).Briefly, nitrocellulose membranes were pre-coated with heat-denaturedhuman collagen I or IV and used to lift E. coli-expressed variant FABsfrom phage plates. The membranes were then incubated with antibodies,either anti-human kappa chain or anti-hemaglutinin (HA) tag conjugatedto alkaline phosphatase to detect bound variant Fabs. Positive cloneswere screened again by single point ELISA (Watkins et al., supra, 1997)for binding to denatured-biotinylated human collagen I and IV,correspondingly. Beneficial variants were characterized for binding toboth collagens in native and heat-denatured forms by ELISA. Beneficialmutations were determined as those having higher affinity binding todenatured collagen relative to the corresponding wild type Fab, asdemonstrated by ELISA.

Shown in FIGS. 4C and 5C is a summary of beneficial CDR mutations in theHUIV26 and HUI77 antibodies, respectively. FIG. 4C summarizes beneficialsingle amino acid mutations in heavy chain CDR1, CDR2, and CDR3 andlight chain CDR1 and CDR3 of HUIV26. An exemplary HUIV26 variant havinga single amino acid substitution is the 12F10Q variant, which exhibitedk_(on) of 0.055 and k_(off) of 0.049 as estimated by the foldimprovement based on shifts in half-maximal binding obtained from ELISAtitrations.

FIG. 5C summarizes beneficial single amino acid mutations in heavy chainCDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of HUI77. As canbe seen, numerous single amino acid mutations in various CDRs were foundto maintain or enhance binding to a cryptic collagen site.

FIG. 5B summarizes beneficial single amino acid mutations in heavy chainCDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of HUI77. As canbe seen, numerous single amino acid mutations in various CDRs were foundto maintain or enhance binding to a cryptic collagen site.

This example describes CDR variants of HUIV26 and HUI77 havingbeneficial mutations.

EXAMPLE III Identification of Combinatorial Variants of HUIV26 and HUI77Antibodes having Enhanced Activity

This example describes the generation and identification ofcombinatorial variants incorporating various beneficial CDR mutations inHUIV26 and HUI77.

To further optimize HUIV26 and HUI77 antibody CDR variants,combinatorial variants, which incorporate at least two CDRs containingone or more mutations, were generated and tested for binding to acryptic collagen site. Combinatorial variants were synthesized usingprimers with one or more positions encoding variant amino acids asdescribed in Example II. The primers used are shown in FIGS. 6 and 7.

Shown in FIGS. 6 and 7 is a summary of the beneficial combinatorialvariants of HUIV26 and HUI77 antibodies, respectively. The k_(on) andk_(off) values shown in FIGS. 6 and 7 (“SPEKon” and “SPEkoff”) wereestimated as the fold improvement of variants based on shifts inhalf-maximal binding obtained from ELISA titrations. Also shown areseveral variants having the same beneficial CDR mutations but havingdifferent framework sequences. These results show that beneficial CDRmutations can be grafted into a variety of frameworks and can retain orhave improved binding activity.

This example shows the generation of combinatorial CDR variants ofHUIV26 and HUI77. A number of variants were identified having increasedaffinity relative to wild type forms of the respective antibodies.

EXAMPLE IV Binding Activity and Specificity of HUIV26 and HUI77 Variants

This example describes the binding activity and specificity of HUIV26and HUI77 antibodies on native and denatured collagen.

The activity and specificity of wild type and selected exemplary HUIV26and HUI77 variants were determined. As shown in FIG. 8, the activity andspecificity of IX-IV26, a Fab containing wild type HUIV26 CDRs, and theHUIV26 variants 2D4H1-C3 and DhuG5 were determined. The antibodies weretested for binding to denatured collagen IV (FIG. 8A), denaturedcollagen I (FIG. 8B), and native collagen IV (FIG. 8C). None of theantibodies had significant binding activity for native collagen IV (FIG.8C). All three antbodies exhibited binding activity for denaturedcollagen IV (FIG. 8A). However, the 2D4H1-C3 and DhuG5 variantsexhibited significantly increased binding activity relative to IX-IV26(FIG. 8A). IX-IV26 did not exhibit significant binding activity todenatured collagen I, and 2D4H1-C3 and DhuG5 exhibited low bindingactivity at the highest measured concentration of antibody (FIG. 8B).These results indicate that the HUIV26 variants have similar bindingactivity and specificity as that of wild type HUIV26 and maintainactivity and specificity for a cryptic collagen epitope. These resultsfurther show that variants having mutated CDRs can have maintained orincreased binding affinity relative to wild type.

As shown in FIG. 9, the activity and specificity of IX-177, a Fabcontaining wild type HUI77 CDRs, and the HUI77 variants Qh2b-B7 andQhuD9 were determined. The antibodies were tested for binding todenatured collagen I (FIG. 9A) denatured collagen IV (FIG. 9B) andnative collagen I (FIG. 9C), and the results indicate that thesevariants exhibited similar binding specificities as wild type. NeitherIX-177 nor Qhu2b-B7 exhibited significant binding activity for nativecollagen I, although the variant QhuD9 exhibited modest binding activityto native collagen at higher concentrations of antibody. The antibodiesall exhibited binding activity for denatured collagen I (FIG. 9A) anddenatured collagen IV (FIG. 9B). However, the Qhu2b-B7 and QhuD9variants exhibited significantly increased binding activity relative toIX-177 on both denatured collagen I and IV. These results indicate thatvariants having mutated CDRs can have maintained or increased bindingaffinity relative to wild type.

To further examine the effect of CDR mutations on binding activity, theHUIV26 variant DhuH8 was selected and expressed in two forms, as a Faband immunoglobulin (IgG). The binding activity of these two forms wasdetermined for native (n-IV) and denatured (d-IV) human collagen IV. Asshown in FIG. 10, neither the Fab nor IgG form of the Dhu8 variantexhibited significant binding to native collagen IV. The Fab formexhibited binding activity for denatured collagen IV, and the bindingaffinity was significantly increased for the IgG form. These resultsindicate that a HUIV26 variant having one or more CDR amino acidsubstitutions relative to wild type can exhibit binding to a crypticcollagen epitope and that the binding affinity can be significantlyincreased in the IgG form relative to the Fab form of the antibodyvariant.

These results indicate that HUIV26 and HUI77 variants having one or moreCDR amino acid substitutions can exhibit similar binding specificity andincreased binding affinity relative to wild type.

EXAMPLE V Generation of Grafted HUIV26 and HUI77 Antibodies HavingOptimized CDRs

This example describes the generation of humanized HUIV26 and HUI77antibodies incorporating beneficial CDR mutations.

A CDR variant have a beneficial mutation is identified as described inExamples II and III. Once a beneficial CDR variant is identified, theCDR variant is grafted into a human framework sequence. In addition tothe CDR variant having a beneficial mutation, other CDRs can be a wildtype sequence of the respective antibody or one or more variant CDRs. Atleast one of the CDRs will be a variant containing a beneficialmutation. For example, if the grafted antibody contains a heavy andlight chain, at least one of the heavy or light chain CDRs will have atleast one amino acid mutation relative to the corresponding wild typeCDR.

A human framework sequence is selected as the recipient for grafting.The human framework can be closely related to the donor antibodyframework sequence or can be relatively divergent from the parentaldonor antibody. Once a human framework is selected for grafting,overlapping oligonucleotides are synthesized encoding the selected humanframework and the appropriate donor CDRs, including at least one variantCDR containing at least one beneficial mutation. The overlappingoligonucleotides are used to assemble a nucleic acid encoding a variableregion including the selected human framework, the CDR variant, andappropriate other CDRs to generate an antibody or fragment havingbinding activity for a cryptic collagen site.

The assembled variable region is cloned into an expression vector, forexample, a Fab expression vector such as described in Example II, andbinding activity to denatured collagen is tested, as described inExamples II and III.

This example describes the generation of humanized antibodies containingbeneficial CDR mutations of HUIV26 and HUI77 antibodies.

EXAMPLE VI Inhibition of B16 Melanoma Cell Proliferation by a VariantHUI77 Antibody

This example describes the effect of the HUI77 variant QH2b on B16melanoma cell proliferation.

The humanized Fab designated QH2b, which is the QH2b-B7 variant of theHUI77 antibody, was engineered into a full length IgG1 antibody(QH2b-IgG1). The QH2b-IgG1 antibody was expressed in mammalian cellculture in NSO cells and purified.

The purified QH2b-IgG1 antibody was used in a cell proliferation assayin vitro. B16 melanoma cells were plated on denatured human Type Icollagen. QH2b-IgG1 (100 μg/ml/day) was added to one set of culturedishes and cell numbers were determined at the indicated times (FIG.11). As a control, the cells were not treated with antibody.

As shown in FIG. 11, B16 melanoma cells proliferated on denaturedcollagen type-I, as indicated by the increase in cell numbers over 3days. The B16 melanoma cell cultures treated with QH2b-IgG1 exhibitedessentially no cell growth over a period of 3 days, indicating that themelanoma cells did not proliferate in the presence of the HUI77 variantQH2b-IgG1.

These results indicate that a HUI77 variant having one or more CDR aminoacid substitutions can inhibit cell proliferation of B16 melanoma cells.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1. An antibody, or antigen-binding fragment thereof, which is a variantof monoclonal antibody HUIV26 having one or more complementaritydetermining regions (CDRs) having an amino acid sequence differing fromthe corresponding CDRs of monoclonal antibody HUIV26, and which hashigher binding affinity for denatured collagen type IV over nativecollagen type IV comprising a heavy chain variable region and a lightchain variable region, wherein said heavy chain variable regioncomprises: (i) a heavy chain CDR1 having the amino acid sequence of SEQID NO: 26 or the amino acid sequence of SEQ ID NO: 26 but for one ormore substitutions selected from the group consisting of: (a)substitution of arginine at position 6 therein by histidine; (b)substitution of methionine at position 9 therein by isoleucine; and (c)substitution of serine at position 10 therein by threonine, alanine orglycine; (ii) a heavy chain CDR2 having the amino acid sequence of SEQID NO: 28 or the amino acid sequence of SEQ ID NO: 28 but for one ormore substitutions selected from the group consisting of: (a)substitution of isoleucine at position 9 therein by alanine, serine orvaline; (b) substitution of serine at position 14 therein by tyro sine,alanine, histidine or glycine; (c) substitution of lysine at position 16therein by aspartic acid or glutamine; and (d) substitution of asparticacid at position 17 therein by lysine or serine; and (iii) a more heavychain CDR3 having the amino acid sequence of SEQ ID NO: 30 or the aminoacid sequence of SEQ ID NO: 30 but for one or substitutions selectedfrom the group consisting of: (a) substitution of aspartic acid atposition 3 therein by proline, glycine, threonine or alanine; (b)substitution of glycine at position 4 therein by proline, alanine orhistidine; and (c) substitution of tyrosine at position 11 therein byproline or asparagine; and wherein said light chain variable regioncomprises: (iv) a light chain CDR1 having the amino acid sequence of SEQID NO: 20 or the amino acid sequence of SEQ ID NO: 20 but for one ormore substitutions selected from the group consisting of: (a)substitution of glutamine at position 4 therein by arginine or serine;(b) substitution of asparagine at position 8 therein by serine; (c)substitution of serine at position 9 therein by tyrosine, tryptophan,histidine or arginine; (d) substitution of glycine at position 10therein by tyrosine, arginine, histidine or isoleucine; and (e)substitution of glutamine at position 12 therein by lysine; (v) a lightchain CDR 2 having the amino acid sequence of SEQ ID NO: 22; and (vi) alight chain CDR3 having the amino acid sequence of SEQ ID NO: 24 or theamino acid sequence of SEQ ID NO: 24 but for one or more substitutionsselected from the group consisting of: (a) substitution of serine atposition 5 therein by glutamine, glycine, leucine, alanine, threonine orvaline; and (b) substitution of tyrosine at position 6 therein byasparagine, serine, proline or methionine.
 2. The antibody orantigen-binding fragment thereof, of claim 1, wherein said heavy chainCDRs are grafted into a VHIII/JH6 heavy chain variable region frameworkreferenced as SEQ ID NO:
 8. 3. The antigen-binding fragment of claim 1,wherein said antigen binding fragment is selected from Fv, Fab, F(ab)₂and scFV fragments.
 4. The antibody or antigen-binding fragment thereof,of claim 1, comprising two of any of said substitutions in the CDRs. 5.The antibody or antigen-binding fragment thereof, of claim 1, comprisingthree of any of said substitutions in the CDRs.
 6. The antibody orantigen-binding fragment thereof, of claim 1, comprising four of any ofsaid substitutions in the CDRs.
 7. The antibody or antigen-bindingfragment thereof, of claim 1, comprising five or more of any of saidsubstitutions in the CDRs.
 8. A nucleic acid encoding an antibody, orantigen binding fragment thereof, of claim
 1. 9. The antibody or antigenbinding fragment thereof of claim 1, wherein said antibody, or antigenbinding fragment thereof further comprises a therapeutic moiety.
 10. Theantibody or antigen binding fragment thereof of claim 1, wherein saidantibody, or antigen binding fragment thereof, further comprises adiagnostic moiety.
 11. A grafted antibody, or antigen-binding fragmentthereof, which is a variant of monoclonal antibody HUIV26 having one ormore complementarity determining regions (CDRs) having an amino acidsequence differing from the corresponding CDRs of monoclonal antibodyHUIV26, and which has higher binding affinity for denatured collagentype IV over native collagen type IV comprising a heavy chain variableregion and a light chain variable region, wherein said heavy chainvariable region comprises: (i) a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 26 or the amino acid sequence of SEQ ID NO: 26but for one or more substitutions selected from the group consisting of:(a) substitution of arginine at position 6 therein by histidine; (b)substitution of methionine at position 9 therein by isoleucine; and (c)substitution of serine at position 10 therein by threonine, alanine orglycine; and (ii) a heavy chain CDR2 having the amino acid sequence ofSEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 28 but for one ormore substitutions selected from the group consisting of: (a)substitution of isoleucine at position 9 therein by alanine, serine orvaline; (b) substitution of serine at position 14 therein by tyrosine,alanine, histidine or glycine; (c) substitution of lysine at position 16therein by aspartic acid or glutamine; and (d) substitution of asparticacid at position 17 therein by lysine or serine; and (iii) a heavy chainCDR3 having the amino acid sequence of SEQ ID NO: 30 or the amino acidsequence of SEQ ID NO: 30 but for one or more substitutions selectedfrom the group consisting of: (a) substitution of aspartic acid atposition 3 therein by proline, glycine, threonine or alanine; (b)substitution of glycine at position 4 therein by proline, alanine orhistidine; and (c) substitution of tyrosine at position 11 therein byproline or asparagine; and wherein said light chain variable regioncomprises: (iv) a light chain CDR1 having the amino acid sequence of SEQID NO: 20 or the amino acid sequence of SEQ ID NO: 20 but for one ormore substitutions selected from the group consisting of: (a)substitution of glutamine at position 4 therein by arginine or serine;(b) substitution of asparagine at position 8 therein by serine; (c)substitution of serine at position 9 therein by tyrosine, tryptophan,histidine or arginine; (d) substitution of glycine at position 10therein by tyrosine, arginine, histidine or isoleucine; and (e)substitution of glutamine at position 12 therein by lysine; and (v) alight chain CDR2 having the amino acid sequence of SEQ ID NO: 22; and(vi) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 24or the amino acid sequence of SEQ ID NO: 24 but for one or moresubstitutions selected from the group consisting of: (a) substitution ofserine at position 5 therein by glutamine, glycine, leucine, alanine,threonine or valine; and (b) substitution of tyrosine at position 6therein by asparagine, serine, proline or methionine.
 12. The graftedantibody or antigen-binding fragment thereof of claim 11, wherein theheavy chain CDRs are grafted into a VHIII/JH6 heavy chain variableregion framework referenced as SEQ ID NO:
 8. 13. The antigen-bindingfragment of claim 11, wherein said antigen binding fragment is selectedfrom Fv, Fab, F(ab)₂ and scFV fragments.
 14. A nucleic acid encoding anantibody or antigen binding fragment thereof of claim
 11. 15. Thegrafted antibody or antigen binding fragment thereof of claim 11,wherein said grafted antibody, or antigen binding fragment thereof,further comprises a therapeutic moiety.
 16. The grafted antibody orantigen binding fragment thereof of claim 11, wherein said graftedantibody, or antigen binding fragment thereof, further comprises adiagnostic moiety.
 17. The grafted antibody or antigen-binding fragmentthereof, of claim 11, comprising two of any of said substitutions in theCDRs.
 18. The grafted antibody or antigen-binding fragment thereof, ofclaim 11, comprising three of any of said substitutions in the CDRs. 19.The grafted antibody or antigen-binding fragment thereof, of claim 11,comprising four of any of said substitutions in the CDRs.
 20. Thegrafted antibody or antigen-binding fragment thereof, of claim 11,comprising five or more of any of said substitutions in the CDRs. 21.The antibody, or antigen-binding fragment thereof, of claim 1 whereinsaid antibody, or antigen-binding fragment thereof, comprises a heavychain CDR1 referenced as SEQ ID NO:45; a heavy chain CDR2 referenced asSEQ D NO:155; a heavy chain CDR3 referenced as SEQ ID NO:63; a lightchain CDR1 referenced as SEQ ID NO: 157; a light chain CDR2 referencedas SEQ ID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.